JoVE General
Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer
Department of Chemistry and Biochemistry, Southern Illinois University
We demonstrate FRET between conjugated polymer polydiacetylene (PDA) and fluorophore attached to the surface of PDA liposomes for the sensing of biomolecules. PDA liposomes also contained receptor molecules on their surfaces for biomolecules to be used as probes. Ligand-receptor interactions lead to changes in the FRET efficiency between the fluorophore and PDA which is the basis of the sensing mechanism.
JoVE Bioengineering
Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells
1Department of Cell Biology, Center for Cell Dynamics, Johns Hopkins University, 2Graduate School of Pharmaceutical Sciences, University of Tokyo, 3Biomedical Engineering, Johns Hopkins University
A method for spatio-temporal control of small GTPase activity by light is described. This method is based on rapamycin-induced FKBP-FRB heterodimerization and photo-caging systems. Optimization of light-irradiation enables the spatio-temporally controlled activation of small GTPases at the subcellular level.
JoVE General
In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy
1Department of Physics, University of Wisconsin - Milwaukee, 2Department of Biological Sciences, University of Wisconsin - Milwaukee
By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of Förster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.
JoVE General
Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum
Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.
JoVE Immunology and Infection
Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with β-arrestin
Microbiology and Immunology, James Graham Brown Cancer Center, University of Louisville
This paper describes the methodology to determine the chemotactic response of leukocytes to specific ligands and identify interactions between the cell surface receptors and cytosolic proteins using live cell imaging techniques.
JoVE General
Imaging Protein-protein Interactions in vivo
Biochemistry and Molecular Biology, Virginia Commonwealth University
This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.
JoVE General
Bimolecular Fluorescence Complementation
Department of Pharmacology, University of Illinois at Chicago
The subcellular localization of proteins is important in determining the spatio-temporal regulation of cell signaling. Here, we describe bimolecular fluorescence complementation (BiFC) as a straightforward method for monitoring the spatial interactions of proteins in the cell.
JoVE General
FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors
Förster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.
JoVE General
Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time
1Mechanical Engineering, Johns Hopkins University, 2Biomedical Engineering, Duke University, 3Biomedical Engineering, Johns Hopkins University
We present a novel and powerful integration of nanophotonics (QD-FRET) and microfluidics to investigate the formation of polyelectrolyte polyplexes, which is expected to provide better control and synthesis of uniform and customizable polyplexes for future nucleic acid-based therapeutics.
JoVE Bioengineering
Quantitative FRET (Förster Resonance Energy Transfer) Analysis for SENP1 Protease Kinetics Determination
Department of Bioengineering, Bourns College of Engineering, University of California, Riverside
A novel method involving quantitative analysis of FRET (Förster Resonance Energy Transfer) signals is described for studying enzyme kinetics. KM and kcat were obtained for the hydrolysis of the catalytic domain of SENP1 (SUMO/Sentrin specific protease 1) to pre-SUMO1 (Small Ubiquitin-like MOdifier). The general principles of this quantitative-FRET-based protease kinetic study can be applied to other proteases.
JoVE General
Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules
1Physics Department, Boston University, 2Department of Biomedical Engineering, Boston University
In this article we describe how we obtain FRET traces from individual DNA molecules immobilized to a surface using an automated scanning confocal microscope.
JoVE General
Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET
Department of Cell and Molecular Biology, Karolinska Institutet
FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.
JoVE General
Measuring Peptide Translocation into Large Unilamellar Vesicles
1Department of Chemistry, Wellesley College,
This protocol details a method for the quantitative measure of peptide translocation into large unilamellar lipid vesicles. This method also provides information about the rate of membrane translocation and can be used to identify peptides that efficiently and spontaneously cross lipid bilayers.
JoVE Neuroscience
In vivo Neuronal Calcium Imaging in C. elegans
1Department of Physiology and Biophysics, Boston University School of Medicine, 2Boston University Photonics Center
With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.
JoVE General
Heterokaryon Technique for Analysis of Cell Type-specific Localization
Department of Chemistry and Biochemistry, Worcester Polytechnic Institute- WPI
A flexible and efficient method for the characterization of cell type-specific protein localization and nucleocytoplasmic shuttling is described. This heterokaryon approach uses fluorescently-labeled fusion proteins to image protein localizations after cell fusion. The protocol is amenable to steady-state localizations or more dynamic determinations based on live cell imaging.
JoVE General
Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems
Department of Biochemistry and Biophysics, University of Pennsylvania
We present principles of oxygen measurements by phosphorescence quenching and review design of porphyrin-based dendritic nanosensors for oxygen imaging in biological systems.
JoVE General
Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling
Department of Chemistry and Biochemistry, Worcester Polytechnic Institute
We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.
JoVE General
Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay
Medical Research Council, Clinical Sciences Centre, Imperial College, Hammersmith Hospital
Here we show how to use Proximity Ligation Assay (PLA), with a combination of antibodies to visualize Bone Morphogenetic Protein (BMP) signaling in fixed cells. This technique allowed us to follow the nuclear accumulation of endogenous BMP activated effector-complexes and quantify their levels over time under BMP4 stimulation.
JoVE General
Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons
1Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, 2Hillman Cancer Center, University of Pittsburgh Cancer Institute, 3Department of Experimental Therapy, The Netherlands Cancer Institute, 4Department of Human Genetics, University of Pittsburgh School of Public Health
We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates. The assay yields rates of DNA Repair activity amenable to kinetic analysis and is adaptable for quantification of DNA Repair activity in tissue and tumor lysates or with purified proteins.
JoVE Bioengineering
Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip
1Department of Plant Biology, Carnegie Institution for Science, 2Howard Hughes Medical Institute, 3Departments of Applied Physics and Bioengineering, Stanford University, 4Department of Microsystems Engineering (IMTEK) and Center for Biological Signaling Studies (BIOSS), University of Freiburg
This article provides a protocol for cultivation of Arabidopsis seedlings in the RootChip, a microfluidic imaging platform that combines automated control of growth conditions with microscopic root monitoring and FRET-based measurement of intracellular metabolite levels.
JoVE Editorial
The 2009 Lindau Nobel Laureate Meeting: Roger Y. Tsien, Chemistry 2008
American biochemist Roger Tsien shared the 2008 Nobel Prize in Chemistry with Martin Chalfie and Osamu Shimomura for their discovery and development of the Green Fluorescent Protein (GFP). Tsien dramatically improved the wild-type GFP resulting in increased fluorescence, increased photostability, and a shift in the major excitation peak to 488 nm (matching FITC).
JoVE Bioengineering
In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage
1Neurosurgery, Baylor College of Medicine, 2Michael E. DeBakey Veterans Affairs Medical Center, 3Molecular & Cellular Biology, Baylor College of Medicine
We demonstrate the assembly and application of a molecular-scale device powered by a topoisomerase protein. The construct is a bio-molecular sensor which labels two major types of DNA breaks in tissue sections by attaching two different fluorophores to their ends.
JoVE Applied Physics
Simulation, Fabrication and Characterization of THz Metamaterial Absorbers
School of Engineering, University of Glasgow
This protocol outlines the simulation, fabrication and characterization of THz metamaterial absorbers. Such absorbers, when coupled with an appropriate sensor, have applications in THz imaging and spectroscopy.
JoVE General
Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering (CARS)
1Center for Biophotonics, University of California, Davis, 2Department of Internal Medicine, University of California, Davis
A combination of three single wavelength short-pulsed lasers is used to generate coherent anti-Stokes Raman scattering (CARS) and doubly-resonant CARS (DR-CARS). The difference between these signals provides enhanced sensitivity for otherwise difficult to detect coherent Raman signals, enabling imaging of weak Raman scatterers.
JoVE Bioengineering
Microfluidic Mixers for Studying Protein Folding
1Department of Physics and Astronomy, Michigan State University, 2Department of Mechanical Engineering, Hong Kong University of Science and Technology, 3Center for Biophotonics, University of California, Davis
In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 μs. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).
JoVE Neuroscience
Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration
Department of Biology, University of Victoria
We have developed a novel technique of quantifying nicotinic acetylcholine receptor changes within subcellular regions of specific subtypes of CNS neurons to better understand the mechanisms of nicotine addiction by using a combination of approaches including fluorescent protein tagging of the receptor using the knock-in approach and spectral confocal imaging.
JoVE General
Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein
Here we describe an optimized technique to produce high-quality vitamin A/RBP complex and two real-time monitoring techniques to study vitamin A transport by STRA6, the RBP receptor.
JoVE Bioengineering
Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria
1Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University, 2Clinical Research Laboratory, 81st Medical Group, Keesler Air Force Base
Lytic phage biosensors and antibody beads are able to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. The phages were immobilized by a Langmuir-Blodgett method onto a surface of a quartz crystal microbalance sensor and worked as broad range staphylococcus probes. Antibody beads recognize MRSA.
JoVE Immunology and Infection
Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs
1Department of Biomedical Engineering, Vanderbilt University, 2Vanderbilt Institute for Nanoscale Science & Engineering, Vanderbilt University, 3Interdisciplinary Materials Science Program, Vanderbilt University, 4Monroe Carell Jr. Children's Hospital, Vanderbilt University Medical Center, 5Department of Chemical & Biomolecular Engineering, Vanderbilt University, 6Department of Cancer Biology, Vanderbilt University
A hemolysis assay can be used as a rapid, high-throughput screen of drug delivery systems' cytocompatibility and endosomolytic activity for intracellular cargo delivery. The assay measures the disruption of erythrocyte membranes as a function of environmental pH.
JoVE Bioengineering
Graphene Coatings for Biomedical Implants
1Department of Physics, Clemson University, 2Department of Pharmacology and Toxicology, East Carolina University, 3Department of Bioengineering, Clemson University, 4Center for Optical Materials Science and Engineering Technologies, Clemson University
Graphene offers potential as a coating material for biomedical implants. In this study we demonstrate a method for coating nitinol alloys with nanometer thick layers of graphene and determine how graphene may influence implant response.
JoVE Immunology and Infection
Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens
1Dynamique des Interactions Hôte Pathogène, Institut Pasteur, Paris, France, 2Imagopole, Institut Pasteur, Paris, France, 3Pathogenomique Mycobacterienne Integrée, Institut Pasteur, Paris, France
We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.
JoVE Neuroscience
Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model
Klinik und Poliklinik für Anästhesiologie, University of Wurzburg
This method describes how to isolate and immortalize microvascular endothelial cells from mouse brain. We describe a step-by-step protocol starting from the homogenization of brain tissue, digestion steps, seeding and immortalization of the cells. Usually, it takes about five weeks to obtain a homogenous, immortalized microvascular endothelial cell line.
JoVE General
Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels
1Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, 2Department of Anesthesiology, David Geffen School of Medicine, University of California, Los Angeles, 3Department of Anesthesiology, Medicine and Physiology, David Geffen School of Medicine, University of California, Los Angeles
We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.
JoVE Applied Physics
Molecular Beam Mass Spectrometry With Tunable Vacuum Ultraviolet (VUV) Synchrotron Radiation
Chemical Sciences Division, Lawrence Berkeley National Laboratory
A molecular beam coupled to tunable vacuum ultraviolet photoionization mass spectrometer at a synchrotron provides a convenient tool to explore the electronic structure of isolated gas phase molecules and clusters. Proton transfer mechanisms in DNA base dimers were elucidated with this technique.
JoVE Bioengineering
Photoacoustic Cystography
1Department of Biomedical Engineering, University at Buffalo, The State University of New York, 2Department of Creative IT Engineering, Pohang University of Science and Technology (POSTECH), 3School of Electrical Engineering and Computer Science, Kyungpook National University
Photoacoustic cystography (PAC) has a great potential to map urinary bladders, a radiation sensitive internal organ in pediatric patients, without using any ionizing radiation or toxic contrast agent. Here we demonstrate the use of PAC for mapping urinary bladders with an injection of optical-opaque tracers in rats in vivo.
JoVE General
Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping
Schulich Faculty of Chemistry, The Technion, Israel Institute of Technology
This protocol describes a method for micron-scale three-dimensional imaging of oxygen concentration in the immediate environment of live cells by electron spin resonance microscopy.
JoVE Bioengineering
Demonstrating the Uses of the Novel Gravitational Force Spectrometer to Stretch and Measure Fibrous Proteins
Department of Biological Sciences, University of North Texas
This is a step-by step guide showing the purpose, operation, and representative results from the novel gravitational force spectrometer.
JoVE Applied Physics
Synthesis and Functionalization of Nitrogen-doped Carbon Nanotube Cups with Gold Nanoparticles as Cork Stoppers
Department of Chemistry, University of Pittsburgh
We discussed the synthesis of individual graphitic nanocups using a series of techniques including chemical vapor deposition, acid oxidation and probe-tip sonication. By citrate reduction of HAuCl4, the graphitic nanocups were effectively corked with gold nanoparticles due to the chemically reactive edges of the cups.
JoVE General
Isolation and Characterization of RNA-Containing Exosomes
This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.
JoVE Bioengineering
Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth
1Department of Biological Systems Engineering, University of Nebraska-Lincoln, 2Department of Engineering Mechanics, University of Nebraska-Lincoln
The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (μMRE).
JoVE Applied Physics
Nanomoulding of Functional Materials, a Versatile Complementary Pattern Replication Method to Nanoimprinting
1Institute of Microengineering (IMT), Photovoltaics and Thin Film Electronics Laboratory, Ecole Polytechnique Fédérale de Lausanne (EPFL), 2Department of Electrical Engineering and Computer Sciences, University of California, Berkeley
We describe a nanomoulding technique which allows low-cost nanoscale patterning of functional materials, materials stacks and full devices. Nanomoulding can be performed on any nanoimprinting setup and can be applied to a wide range of materials and deposition processes.
JoVE Applied Physics
Angle-resolved Photoemission Spectroscopy At Ultra-low Temperatures
1Institute for Solid State Research, IFW-Dresden, 2Institute of Metal Physics of National Academy of Sciences of Ukraine, 3Diamond Light Source LTD, 4Department of Physics, University of Johannesburg, 5CNR-SPIN, and Dipartimento di Fisica "E. R. Caianiello", Università di Salerno, 6Institute of Physics of Complex Matter, École Polytechnique Fédérale de Lausanne
The overall goal of this method is to determine the low-energy electronic structure of solids at ultra-low temperatures using Angle-Resolved Photoemission Spectroscopy with synchrotron radiation.
JoVE Bioengineering
Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry
1Biomedical Engineering Department, Johns Hopkins University School of Medicine, 2Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, 3Wilmer Eye Institute, Johns Hopkins University School of Medicine, 4Institute for Nanobiotechnology, Johns Hopkins University School of Medicine
A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.
JoVE Clinical and Translational Medicine
Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy
Department of Anesthesiology & Pain Medicine, University of Washington School of Medicine
Langendorff-mode isolated heart perfusion, in conjunction with 31P NMR spectroscopy, combines the fields of biochemistry and physiology into one experiment. The protocol allows for the dynamic measurement of high energy phosphate content and turnover in the heart while concurrently monitoring physiologic function. When performed correctly, this is a valuable technique in the assessment of cardiac energetics.
JoVE Bioengineering
Synthetic, Multi-Layer, Self-Oscillating Vocal Fold Model Fabrication
Department of Mechanical Engineering, Brigham Young University
The methodology for fabricating synthetic vocal fold models is described. The models are life-sized and mimic the multi-layer structure of the human vocal folds. Results show the models to self-oscillate at pressures comparable to lung pressure and demonstrate flow-induced vibratory responses that are similar to those of human vocal folds.
JoVE General
Determining the Contribution of the Energy Systems During Exercise
1Laboratory of Applied Nutrition, School of Physical Education and Sport, University of Sao Paulo, 2Aerobic Performance Research Group, School of Physical Education and Sport, University of Sao Paulo, 3Laboratory of Neuromuscular Adaptations to Strength Training, School of Physical Education and Sport, University of Sao Paulo, 4Martial Arts and Combat Sports Research Group, School of Physical Education and Sport, University of Sao Paulo
This protocol allows researchers focused on exercise and sports sciences to determine the relative contribution of three different energy systems to the total energy expenditure during a large variety of exercises.
JoVE General
Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System
1Department of Biology, University of Western Ontario, 2Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada
We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.
JoVE Immunology and Infection
Customization of Aspergillus niger Morphology Through Addition of Talc Micro Particles
Institute of Biochemical Engineering, Technische Universität Braunschweig
A method to precisely generate and to comprehensively characterize morphology of filamentous fungus Aspergillus niger is described, which allows the mathematical correlation of morphological appearance and productivity.
JoVE Applied Physics
Synthesis and Operation of Fluorescent-core Microcavities for Refractometric Sensing
Department of Physics, University of Alberta
Fluorescent-core microcavity sensors employ a high-index quantum-dot coating in the channel of silica microcapillaries. Changes in the refractive index of fluids pumped into the capillary channel cause shifts in the microcavity fluorescence spectrum that can be used to analyze the channel medium.
JoVE Applied Physics
Hyperpolarized Xenon for NMR and MRI Applications
ERC Project BiosensorImaging, Leibniz-Institut für Molekulare Pharmakologie
The production of hyperpolarized xenon by means of spin exchange optical pumping (SEOP) is described. This method yields a ~10000-fold enhancement of the nuclear spin polarization of Xe-129 and has applications in nuclear magnetic resonance spectroscopy and imaging. Examples of gas phase and solution state experiments are given.
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