In this article, we present a microfluidic-based method for particle confinement based on hydrodynamic flow. We demonstrate stable particle trapping at a fluid stagnation point using a feedback control mechanism, thereby enabling confinement and micromanipulation of arbitrary particles in an integrated microdevice.
1Department of Medicine, Division of Infectious Diseases, Massachusetts General Hospital, Harvard Medical School, 2Department of Mechanical and Aerospace Engineering, The Ohio State University, 3Center for Computational and Integrative Biology, Massachusetts General Hospital, Harvard Medical School, 4Dept. of Chemical and Biomolecular Engineering, Vanderbilt University
A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.
Spin-trapping ESR spectroscopy was used to study the effect of plant antioxidants lycopene, pycnogenol and grape seed extract on scavenging gas-phase free radicals in cigarette smoke.
Construction of a High Resolution Microscope with Conventional and Holographic Optical Trapping Capabilities
The system described herein employs a traditional optical trap as well as an independent holographic optical trapping line, capable of creating and manipulating multiple traps. This allows for the creation of complex geometric arrangements of refractive particles while also permitting simultaneous high-speed, high-resolution measurements of the activity of biological enzymes.
Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation
1Electrical Engineering Department, University of Washington, 2Division of Human Biology, Fred Hutchinson Cancer Research Center, 3Molecular and Cellular Biology Program, University of Washington, 4Clinical Research, Fred Hutchinson Cancer Research Center, 5Public Health Sciences, Fred Hutchinson Cancer Research Center
Plasmonic tweezers and photonic crystal nanostructures are shown to produce useful enhancements in the efficiency and orientation control of optically trapping micro- and nano-particles.
Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).
The following setup approach details low power optical trapping of dielectric nanoparticles using a double-nanohole in metal film.
We illustrate the use of a constant force axial optical tweezers to explore the mechanical properties of short DNA molecules. By stretching DNA axially, we minimize steric hindrances and artifacts arising in conventional lateral manipulation, allowing us to study DNA molecules as short as ~100 nm.
This video describes the manipulation of cultured neurons using laser tweezers in vitro.
We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.
Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps
Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.
1Undergraduate Program, Rice University, 2Proteomics Facility, Department of Pathology, University of Texas MD Anderson Cancer Center, 3Department of Melanoma Medical Oncology, University of Texas MD Anderson Cancer Center, 4University of Texas Graduate School of Biological Sciences at Houston
A specific and sensitive method to gain insight into the expression profile of glycosphingolipid antigens in immune organs and cells is described. The method takes advantage of the ion trap mass spectrometry allowing step-wise fragmentation of glycosphingolipid molecules for structural analysis in comparison to chemically synthesized standards.
Ion mobility-mass spectrometry is an emerging gas-phase technology that separates ions, based on their collision cross-section and mass. The method provides three-dimensional information on the overall topology and shape of protein complexes. Here, we outline a basic procedure for instrument setting and optimization, calibration of drift times, and data interpretation.
Neutrophil Extracellular Traps (NETs) are an important innate immune mechanism to fight pathogenic bacteria, fungi and parasites. Here we describe methods to isolate neutrophil granulocytes from human blood and to activate them to form NETs. We present preparation techniques to visualize NETs in light and electron microscopy.
Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana
The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.
Behavior assays for measuring locomotor functions, learning, and memory abilities in Drosophila.
A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.
Perfusion decellularization is a novel technique to produce whole liver scaffolds that retains the organ's extracellular matrix composition and microarchitecture. Herein, the method of preparing whole organ scaffolds using perfusion decellularization and subsequent repopulation with hepatocytes is described. Functional and transplantable liver grafts can be generated using this technique.
Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy
We show, how to use 2-photon microscopy for the observation of the dynamics of neutrophil granulocytes in infected lungs while they phagocytose pathogens or produce neutrophil extracellular traps (NETs).
Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines
1Department of Biology, Pace University, 2Cellular and Molecular Medicine, University of California, San Diego, 3Division of Cell Biology and Cell Physiology, Zoological Institute, Braunschweig University of Technology
The temporal and spatial resolution of genetic manipulations determines the spectrum of biological phenomena that they can perturb. Here we use temporally and spatially discrete in vivo electroporation, combined with transgenic lines of zebrafish, to induce expression of a GFP transgene specifically in neurons of the developing olfactory bulb.
Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
1Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California at Los Angeles, 2Crump Institute for Molecular Imaging, David Geffen School of Medicine, University of California at Los Angeles, 3California NanoSystems Institute, University of California at Los Angeles, 4Nuclear Medicine, PET Center, Shanghai Medical Collegea, Fudan University, 5Electronics and Information Engineering, College of Electronics and Information Engineering, Wuhan Textile University
A facile, one-pot synthesis of N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) was developed based on a non-aqueous, three-step radiochemical process. Using microwave heating, the entire procedure can be completed in less than 30 min, or 60 min with further purification by preparative HPLC. The decay-corrected radiochemical yields (RCYs) were 35-5% (n > 30).
Continuously-stirred Anaerobic Digester to Convert Organic Wastes into Biogas: System Setup and Basic Operation
Laboratory-scale anaerobic digesters allow scientists to research new ways of optimizing existing applications of anaerobic biotechnology and to evaluate the methane producing potential of various organic wastes. This article introduces a generalized model for the construction, inoculation, operation, and monitoring of a laboratory-scale continuously stirred anaerobic digester.
Sampling Human Indigenous Saliva Peptidome Using a Lollipop-Like Ultrafiltration Probe: Simplify and Enhance Peptide Detection for Clinical Mass Spectrometry
Considering saliva sampling for future clinical application, a lollipop-like ultrafiltration (LLUF) probe was fabricated to fit in the human oral cavity. Direct analysis of undigested saliva by NanoLC-LTQ mass spectrometry demonstrated the ability of LLUF probes to remove large proteins and high abundance proteins, and make low-abundant peptides more detectable.
Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.
Avian Influenza Surveillance with FTA Cards: Field Methods, Biosafety, and Transportation Issues Solved
A method to preserve, detect and sequence RNA from Avian Influenza Viruses was validated and extended using natural faecal samples from birds. This technique removes the necessity of maintaining a cool chain and handling of infectious viruses and can be applied in a 96-well high-throughput setup.
This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.
A method for large-scale purification of the APP intracellular domain (AICD) is described. We also describe methodology to induce in vitro AICD aggregation and visualization by atomic force microscopy. The methods described are useful for biochemical/structural characterization of the AICD and the effects of molecular chaperones on its aggregation.
Demonstrating the Uses of the Novel Gravitational Force Spectrometer to Stretch and Measure Fibrous Proteins
This is a step-by step guide showing the purpose, operation, and representative results from the novel gravitational force spectrometer.
Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.
1Department of Anesthesiology, Perioperative and Pain Medicine, Children's Hospital Boston and Harvard Medical School, 2Departments of Cardiac Surgery, Children's Hospital Boston and Harvard Medical School
This article describes a high temporal and spatial resolution technique to optically image action potential movement on the surface of Langendorff-perfused rat hearts using a potentiometric dye (di-8-ANEPPS).
A simple method is provided that allows for the rapid extraction and analysis of multiple plant hormones from small tissue samples. The procedure uses vapor phase extraction as the solemn purification step. Samples are analyzed by GC/MS with chemical ionization that produces mainly (M+1)+ ions.
We have used plasma enhanced chemical vapor deposition to deposit thin films ranging from a few nm to several 100 nm on nano-sized particles of various materials. We subsequently etch the core material to produce hollow nanoshells whose permeability is controlled by the thickness of the shell. We characterize the permeability of these coatings to small solutes and demonstrate that these barriers can provide sustained release of the core material over several days.
Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe
Olfactory cues mediate many different behaviors in insects, and are often complex mixtures comprised of tens to hundreds of volatile compounds. Using gas chromatography with multi-channel recording in the insect antennal lobe, we describe a method for the identification of bioactive compounds.
1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production
Here's a look at some of the milestones and highlights of the year 2012 in Journal of Visualized Experiments (JoVE).
Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.
A push-pull method for collecting plant volatiles is described. The method allows for a comparison of volatiles induced by herbivore feeding, exogenous methyl jasmonate, and mechanical damage. This technique is also used to investigate the volatile response of undamaged branches to exposure to volatiles from herbivore-damaged branches within blueberry plants.
In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.
1Center for Proteomics, Smith College, 2Department of Molecular Biophysics and Biochemistry, Yale University, 3Department of Chemistry, Smith College, 4Department of Biological Sciences and Center for Proteomics, Smith College
A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues.
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
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.
We describe a Flippase-induced intersectional Gal80/Gal4 repression (FINGR) method, allowing tissue-specific FLP to determine Gal80 expression patterns. Wherever Gal4 and FLP overlap, Gal4 expression is turned on (Gal80 flipped out) or off (Gal80 flipped in). The FINGR method is versatile for clonal analysis and neural circuit mapping.
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.
An automated microfluidic device was developed for circulating nucleated cell enrichment from peripheral blood via erythrocyte lysis that ensures isolation of high quality sample without cell loss.
This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.
Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease
Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.
We describe the preparation of colloidal quantum dots with minimized hydrodynamic size for single-molecule fluorescence imaging. Compared to conventional quantum dots, these nanoparticles are similar in size to globular proteins and are optimized for single-molecule brightness, stability against photodegradation, and resistance to nonspecific binding to proteins and cells.
Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein
Msh2-Msh6 is responsible for initiating repair of replication errors in DNA. Here we present a transient kinetics approach towards understanding how this critical protein works. The report illustrates stopped-flow experiments for measuring the coupled DNA binding and ATPase kinetics underlying Msh2-Msh6 mechanism of action in DNA repair.
Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.
Cutaneous Leishmaniasis in the Dorsal Skin of Hamsters: a Useful Model for the Screening of Antileishmanial Drugs
Optimization of the experimental hamster model for cutaneous leishmaniasis by intradermal injection of Leishmania promastigotes at the dorsal skin. This approach is useful during inoculation, follow-up, characterization of lesions, application of treatments and obtaining of clinical samples. Locomotion, search for food and water, play and social activities are preserved.
The IonFlux Automated Patch Clamp System provides high throughput, cost-effective ion channel screening for a wide range of electrophysiology applications. Fast compound exchange, low cost per data point, and convenient well plate formats make the system ideal for both ligand- and voltage-gated ion channel targets. The IonFlux HT provides an industry-leading 10,000 data points per day, while the IonFlux 16 provides true automated patch clamp performance for about the cost of a manual patch clamp rig.