Near Infrared Optical Projection Tomography for Assessments of β-cell Mass Distribution in Diabetes Research
1Umeå Centre for Molecular Medicine, Umeå University, 2Cell Transplant Center, Diabetes Research Institute, University of Miami,, 3EMBL-CRG Systems Biology Program, Centre for Genomic Regulation, Catalan Institute of Research and Advanced Studies, 4Dept. of Computing Science, Umeå University
We describe the adaptation of optical projection tomography (OPT)1 to imaging in the near infrared spectrum, and the implementation of a number of computational tools. These protocols enable assessments of pancreatic β-cell mass (BCM) in larger specimens, increase the multichannel capacity of the technique and increase the quality of OPT data.
Here we describe a protocol for the preparation of agar-embedded retinal slices that are suitable for electrophysiology and Ca2+ imaging. This method allows one to study ribbon-type synapses in retinal microcircuits using direct patch-clamp recordings of single presynaptic nerve terminals.
Here are some highlights from the December 2011 Issue of Journal of Visualized Experiments (JoVE).
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.
We present a Turing-like Handshake test administered through a telerobotic system in which the interrogator is holding a robotic stylus and interacting with another party (human or artificial). We use a forced choice method, and extract a measure for the similarity of the artificial model to a human handshake.
1Department of Biostatistics, Virginia Commonwealth University, 2Virginia Commonwealth University Reanimation Engineering Science (VCURES) Center, 3Department of Computer Science, Virginia Commonwealth University, 4Department of Radiology, Virginia Commonwealth University, 5Department of Emergency Medicine, Virginia Commonwealth University
An automated midline shift estimation and intracranial pressure (ICP) pre-screening system based on computed tomography (CT) images for patients with traumatic brain injury (TBI) is proposed using image processing and machine learning techniques.
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.
1Brain and Behavior Discovery Institute, Georgia Health Sciences University, 2Vision Discovery Institute, Georgia Health Sciences University, 3Department of Opthalmology, Georgia Health Sciences University, 4Intelligent Systems Laboratory, Palo Alto Research Center, 5Pattern Recognition Systems, Palo Alto Research Center, 6Department of Psychology, University of Minnesota
We describe a novel methodology for creating naturalistic 3-D objects and object categories with precisely defined feature variations. We use simulations of the biological processes of morphogenesis and phylogenesis to create novel, naturalistic virtual 3-D objects and object categories that can then be rendered as visual images or haptic objects.
Combining Computer Game-Based Behavioural Experiments With High-Density EEG and Infrared Gaze Tracking
1Department of Human Development, Cornell University, 2Social Sciences Division, University of Chicago, 3National Brain Research Centre, Manesar, India
Procedures for recording high-density EEG and gaze data during computer game-based cognitive tasks are described. Using a video game to present cognitive tasks enhances ecological validity without sacrificing experimental control.
Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging
1Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 2The Medical Research Council Mitochondrial Biology Unit, University of Cambridge, 3National Library of Medicine, National Institutes of Health, 4Massachusetts Institute of Technology, 5William Fremd High School, 6University of Virginia, 7Duke University, 8Yale University, 9University of Notre Dame, 10Washington University in St. Louis, 11Bioinformatics and Computational Biosciences Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12Thomas Jefferson High School for Science and Technology
The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.
The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.
CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.
Demonstration of key methods for high throughput leaf measurements. These methods can be used to accelerate leaf phenotyping when studying many plant mutants or otherwise screening plants by leaf phenotype.
We describe the design and assembly of miniaturized headphones suitable for replacing a songbird’s natural auditory feedback with a manipulated acoustic signal. Online sound processing hardware is used to manipulate song output, introduce real-time errors in auditory feedback via the headphones, and drive vocal motor learning.
Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence
1Department of Microbiology and Immunology, University of Rochester Medical Center, 2Center for Neural Development and Disease, University of Rochester Medical Center, 3Deptartment of Neurology, Center for Neural Development and Disease, University of Rochester Medical Center
Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.
Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.
Dopamine replacement pharmacotherapy using L-DOPA is the most commonly used symptomatic treatment of Parkinson’s disease, but is accompanied by side effects including involuntary abnormal movements, termed dyskinesia 1. Here, a protocol for MALDI imaging mass spectrometry is presented that detects changes in rat brain neuropeptide levels related to dyskinesia.
1Center for Human Genetic Research and Department of Medicine, Massachusetts General Hospital and Harvard Medical School, 2Department of Earth, Atmospheric, and Planetary Sciences, Massachusetts Institute of Technology
We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.
1Center for the Neural Basis of Cognition, 2Department of Psychology, University of Pittsburgh, 3Department of Psychology, Carnegie Mellon University, 4Department of Bioengineering, University of Pittsburgh
We describe a novel approach for simultaneous analysis of brain function and structure using magnetic resonance imaging (MRI). We assess brain structure with high-resolution diffusion-weighted imaging and white-matter fiber tractography. Unlike standard structural MRI, these techniques allow us to directly relate anatomical connectivity to functional properties of brain networks.
Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS)
1Centre de Recherche de l’Institut du Cerveau et de la Moelle Épinière, Hôpital de la Pitié-Salpêtrière, 2Institut des Sciences Moléculaires d'Orsay, Université Paris-Sud, 3Centre de Photonique Biomédicale du Centre Laser, Université Paris-Sud
A technique to probe the lipid raft partitioning of fluorescent proteins at the plasma membrane of living cells is described. It takes advantage of the disparity in diffusion times of proteins located inside or outside of lipid rafts. Acquisition can be performed dynamically in control conditions or after drug addition.
The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.
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
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.
Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts
We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.
Dorsal Column Steerability with Dual Parallel Leads using Dedicated Power Sources: A Computational Model
Using a mathematical model of spinal cord stimulation, we found that a multi-source system with independent power sources for each contact can target more central points of stimulation on the dorsal column (100 vs 3) and has 50-fold more field steering resolution (0.02mm vs 1mm) than a single-source system.
1Department of Mechanical Engineering, Texas A&M University, 2Department of Mechanical Engineering and Department of Nuclear Engineering, Texas A&M University, 3Department of Chemical Engineering, Texas A&M University
We describe a novel method to perform DNA replication via the polymerase chain reaction (PCR). Thermal convection is harnessed to continuously shuttle reagents between denaturing, annealing, and extension conditions by maintaining opposing surfaces of the reactor at constant temperature. This inherently simple design promises to make rapid PCR more accessible.
The demonstration of the small and wide angle X-ray scattering (SWAXS) procedure has become instrumental in the study of biological macromolecules. Through the use of the instrumentation and procedures of specific angle methods and preparation, the experimental data from the SWAXS displays the atomic and nano-scale characterization of macromolecules.
An echo particle image velocimetry (EPIV) system capable of acquiring two-dimensional fields of velocity in optically opaque fluids or through optically opaque geometries is described, and validation measurements in pipe flow are reported.
Microscopic organisms like the free-swimming nematode C. elegans, live and behave in a complex three-dimensional environment. We report on a novel approach that provides analysis of C. elegans using diffraction patterns. This approach consists of tracking the temporal periodicity of diffraction patterns generated by directing laser light through a cuvette.
A Video Demonstration of Preserved Piloting by Scent Tracking but Impaired Dead Reckoning After Fimbria-Fornix Lesions in the Rat
In a piloting scent tracking task, the ability of the rats to return to a refuge with food using visual an odor trail or using dead reckoning in infrared light, the integrated record of previous movements, demonstrates that the hippocampus is necessary for dead reckoning.
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.
Here we present a histological method for capturing, labeling, optically clearing, and imaging the intact brain tissue interface around chronically implanted microdevices in rodent brain tissue. Results from the techniques comprising this method are useful for understanding the impact of various penetrating brain-implants on their surrounding tissue.
1Department of Computer Science and Engineering, Texas A&M University, 2Beckman Institute for Advanced Science and Technology, University of Illinois, 3Department of Electrical and Computer Engineering, Kettering University, 43Scan, 5Department of Veterinary Integrative Biosciences, Texas A&M University
The full process from brain specimen preparation to serial sectioning imaging using the Knife-Edge Scanning Microscope, to data visualization and analysis is described. This technique is currently used to acquire mouse brain data, but it is applicable to other organs, other species.
1Department of Molecular Genetics, Weizmann Institute of Science, 2Department of Biological Regulation, Weizmann Institute of Science, 3Department of Chemical Infrastructure, Weizmann Institute of Science
Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography ( CT).
Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy. Insight is given into how computational models can provide information on the population dynamics of mosquitoes and the spread of transposable elements through A. gambiae subspecies. The ethical considerations of releasing genetically modified mosquitoes into the wild are discussed.
Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays
1Department of Molecular Genetics, University of Toronto, 2Banting and Best Department of Medical Research, Donnelly Centre, University of Toronto, 3Department of Biochemistry, Research and Innovation Centre, University of Regina
Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.
Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells
MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.
The ITS2 Database is a workbench for phylogenetic inference simultaneously considering sequence and secondary structure of the internal transcribed spacer 2. This includes data collection with accurate annotation, structure prediction, multiple sequence-structure alignment and fast tree calculation. In a nutshell, this workbench simplifies first phylogenetic analyses to a few clicks.
Deep brain stimulation surgery offers a unique opportunity to examine information encoding in the awake human brain. This article will describe intra-operative methods used to perform cognitive and behavioral tasks while simultaneously acquiring physiological data such as EMG, single-unit neuronal activity and/or local field potentials.
How to Measure Cortical Folding from MR Images: a Step-by-Step Tutorial to Compute Local Gyrification Index
1Department of Psychiatry, University of Geneva School of Medicine, 2Signal Processing Laboratory, École Polytechnique Fédérale de Lausanne, 3Department of Radiology, University Hospital Center and University of Lausanne, 4Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital
Measuring gyrification (cortical folding) at any age represents a window into early brain development. Hence, we previously developed an algorithm to measure local gyrification at thousands of points over the hemisphere1. In this paper, we detail the computation of this local gyrification index.
1Australian Centre of Excellence in Plant Cell Walls, School of Botany, University of Melbourne, 2Plant Cell Biology Research Centre, School of Botany, University of Melbourne, 3CSIRO Plant Industry, Black Mountain Laboratories, 4Department of Plant Biology and Biotechnology, University of Copenhagen
A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.
In this video, we demonstrate the experimental techniques used to fabricate compliant, extracellular matrix (ECM) coated substrates suitable for cell culture, and which are amenable to traction force microscopy and observing effects of ECM stiffness on cell behavior.
The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures.
Gene microarrays are powerful tools in gene expression profiling at a genome-wide level. This technology has application in a variety of biological disciplines including developmental biology and toxicology. In this video, we detail a protocol for global gene expression analysis using a comprehensive oligonucleotide microarray platform for the zebrafish.
We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.
Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.
iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution
1Laboratory of Molecular Biology, Medical Research Council - MRC, 2European Bioinformatics Institute, EMBL Heidelberg, 3Computer and Information Science, University of Ljubljana, 4Wellcome Trust Genome Campus, Wellcome Trust Sanger Institute
The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein.
Magnetic resonance imaging (MRI) provides a powerful tool to evaluate the effectiveness of process equipment during operation. We discuss the use of MRI to visualize mixing in a static mixer. The application is relevant to personal care products, but can be applied to a broad range of food, chemical, biomass and biological fluids.
The opener muscle of the crayfish leg is presented for its historical importance and experimental versatility in muscle phenotype, synaptic physiology and plasticity.
Stent-induced arterial strain distributions are characterized using an optical surface strain measurement system. This visualization technique is used to gain insights into the impact of stent implantation on the host vessel.
1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production
Traditional microscopy requires lens objectives to magnify specimens, and can involve numerous optical components like additional objectives, filters, and mirrors to refract and direct light to optical sensors. The August 2012 issue of JoVE (Journal of Visualized Experiments) is marked by the third publication from the Ozcan Lab (University of California, Los Angeles) on their lens-free "on-chip" microscopy platform, which they have pioneered.