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.
MPI CyberMotion Simulator: Implementation of a Novel Motion Simulator to Investigate Multisensory Path Integration in Three Dimensions
1Department of Human Perception, Cognition and Action, Max Planck Institute for Biological Cybernetics, 2Laboratoire de Physiologie de la Perception et de l'Action, Collège de France - CNRS, 3Department of Brain and Cognitive Engineering, Korea University
An efficient way to gain insight into how humans navigate themselves in three dimensions is described. The method takes advantage of a motion simulator capable of moving observers in ways unattainable by traditional simulators. Results confirm that movement in the horizontal plane is underestimated, while vertical movement is overestimated.
Here are some highlights from the May 2012 Issue of Journal of Visualized Experiments (JoVE).
We discuss the construction and operation of a complex nonlinear optical system that uses ultrafast all-optical switching to isolate Raman from fluorescence signals. Using this system we are able to successfully separate Raman and fluorescence signals utilizing pulse energies and average powers that remain biologically safe.
Patterned Photostimulation with Digital Micromirror Devices to Investigate Dendritic Integration Across Branch Points
Digital micromirror devices (DMD) can generate complex patterns in time and space with which to control neuronal excitability. Issues relevant to the design, construction, and operation of DMD systems are discussed. Such a system enabled the demonstration of non-linear integration across distal dendritic branch points.
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.
1Electrical Engineering Department, University of California, Los Angeles, 2Bioengineering Department, University of California, Los Angeles, 3California NanoSystems Institute (CNSI), University of California, Los Angeles
We review our recent results on the integration of fluorescent microscopy and imaging flow cytometry tools on a cell-phone using compact and cost-effective opto-fluidic attachments. These cell-phone based micro-analysis devices might be useful for cytometric analysis, such as performing various cell counting tasks as well as for high-throughput screening of e.g., water samples in resource limited settings.
We describe a super-resolution imaging method to probe the structural organization of the bacterial FtsZ-ring, an essential apparatus for cell division. This method is based on quantitative analyses of photoactivated localization microscopy (PALM) images and can be applied to other bacterial cytoskeletal proteins.
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.
1Banting and Best Department of Medical Research and Department of Molecular Genetics, University of Toronto, 2Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 3Donnelly Sequencing Centre, University of Toronto, 4Genetics and Molecular Biology Branch, National Human Genome Research Institute, NIH, 5Stanford Genome Technology Center, Stanford School of Medicine, Stanford University, 6Department of Pharmaceutical Sciences, University of Toronto
We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.
Development of an Audio-based Virtual Gaming Environment to Assist with Navigation Skills in the Blind
1Laboratory for Visual Neuroplasticity, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, 2Department of Computer Science and Center for Advanced Research in Education (CARE), University of Chile
Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind.
Murine Spinotrapezius Model to Assess the Impact of Arteriolar Ligation on Microvascular Function and Remodeling
1Department of Biomedical Engineering, University of Virginia, 2Department of Biomedical Engineering, California Polytechnic State University, 3Office of Animal Welfare, University of Virginia, 4Department of Biomedical Engineering & Institute for Computational Medicine, Johns Hopkins University
We demonstrate a novel arterial ligation model in murine spinotrapezius muscle, including a step-by-step procedure and description of required instrumentation. We describe the surgery and relevant outcome measurements relating to vascular network remodeling and functional vasodilation using intravital and confocal microscopy.
Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation
This video demonstrates how to visualize axonal pathways of genetically defined groups of neurons in the embryonic chick spinal cord utilizing in ovo electroporation of reporter genes under the control of specific enhancer elements.
1Immunology Graduate Program, Johns Hopkins University, 2Department of Internal Medicine, Far-Eastern Memorial Hospital, 3Department of Pathology, Johns Hopkins University, 4Institute of Cell Engineering, Johns Hopkins University
A new DC independent method for induction and expansion of antigen-specific T cells is described. HLA A2-Ig based artificial Antigen Presenting Cells (aAPC) are loaded with HLA-A2 restricted peptides to efficiently expand CTL of diverse antigen specificity. This technology holds great potential for CTL-based adoptive immunotherapy.
Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging
A method is described for labeling neurons with fluorescent dyes in predetermined functional micro-domains of the neocortex. First, intrinsic signal optical imaging is used to obtain a functional map. Then two-photon microscopy is used to label and image neurons within a micro-domain of the map.
A high-sensitivity photonic micro sensor was developed for electric field detection. The sensor exploits the optical modes of a dielectric sphere. Changes in the external electric field perturb the sphere morphology leading to shifts in its optical modes. The electric field strength is measured by monitoring these optical shifts.
Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster
Phenotypic variation for traits can result from mutations in cis-regulatory element (CRE) sequences that control gene expression patterns. Methods derived for use in Drosophila melanogaster can quantitatively compare the levels of spatial and temporal patterns of gene expression mediated by modified or naturally occurring CRE variants.
A rapid and simple way to generate human cell lines with inducible and reversible cDNA overexpression or shRNA-mediated knock-down of the gene of interest. This method enables researchers to reliably and highly reproducibly manipulate cell lines that are difficult to alter by transient transfection methods or conventional knockdown/knockout strategies.
A way to study the integration of newborn dentate granule cells in adult animals is described. This technique uses an engineered retrovirus to label newborn neurons, followed by electrophysiological recordings to determine in vivo functional integration.
The complete construction of a custom, real-time confocal scanning imaging system is described. This system, which can be readily used for video-rate microscopy and microendoscopy, allows for an array of imaging geometries and applications not accessible using standard commercial confocal systems, at a fraction of the cost.
We demonstrate protocols for manufacturing and automating elastomeric polydimethylsiloxane (PDMS)-based microvalve arrays that need no extra energy to close and feature photolithographically defined precise volumes. A parallel subnanoliter-volume mixer and an integrated microfluidic perfusion system are presented.
Most studies of herpetic corneal disease use a primary infection model. However, primary infection with HSV-1 does not typically lead to human disease. Here we describe a recurrent model of herpetic corneal disease, which more closely mimics human disease.
The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.
MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments. Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables noninvasive and portable monitoring of cerebral blood oxygenation changes. This paper summarizes collective use of MazeSuite and fNIR within a cognitive processing learning paradigm.
TransFLP — A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination
A quick method to modify the genome of V. cholerae is described. These modifications include the deletion of single genes, gene clusters and genomic islands as well as the integration of short sequences (e.g. promoter elements or affinity-tag sequences). The method is based on the natural transformation and FLP-recombination.
Computed Tomography-guided Time-domain Diffuse Fluorescence Tomography in Small Animals for Localization of Cancer Biomarkers
1Thayer School of Engineering, Dartmouth College, 2Department of Physics and Astronomy, Dartmouth College, 3Darmouth Medical School, Dartmouth College, 4School of Computer Science, University of Birmingham
Diffuse fluorescence tomography offers a relatively low-cost and potentially high-throughout approach to preclinical in vivo tumor imaging. The methodology of optical data collection, calibration, and image reconstruction is presented for a computed tomography-guided non-contact time-domain system using fluorescent targeting of the tumor biomarker epidermal growth factor receptor in a mouse glioma model.
We have developed a flow cytometer using laser induced ultrasound to detect circulating melanoma cells as an early indicator of metastatic disease.
Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model
1Department of Orthopaedic Sports Medicine, Klinikum rechts der Isar der Technischen Universität München, 2Department of Radiology, Klinikum rechts der Isar der Technischen Universität München, 3Institute of Experimental Oncology and Therapy Research, Klinikum rechts der Isar der Technischen Universität München, 4Department of Radiology, Uniklinik Köln
An experimental technique for the treatment of chondral defects in the rabbit's knee joint is described. The implantation of autologous chondrocytes seeded on a matrix is a well-accepted method for the remodeling and repair of articular cartilage lesions providing satisfying long-term results. Matrix-assisted autologous chondrocyte transplantation (MACT) offers a standardized and clinically established implantation method.
Determination of gastric emptying with a non-invasive [13C]-octanoic acid breath test for tracking gastroparesis in female NOD LtJ mice.
This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.
1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production
Here are some highlights from the April 2013 Issue of Journal of Visualized Experiments (JoVE).
This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.
We demonstrate a minimally invasive technique referred to as neonatal subventricular zone electroporation. The technique consists of injecting plasmid DNA into the lateral ventricles of neonatal pups and applying electrical current to deliver and genetically manipulate neural stem cells
This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.
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.
Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.
A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.
The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.
The Morris Water Maze is a behavioral task to test hippocampal-dependent learning and memory. It has been widely used in the study of neurobiology, neuropharmacology and neurocognitive disorders in rodent models.
This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.
The inoculation of Trypanosoma cruzi in fertile eggs prior to incubation renders the parasite kDNA minicircle integration in embryo cells genome. Crossbreeding reveals the vertical transfer of the mutations to progeny. The kDNA integrates into coding regions at several chromosomes and the chickens die with an inflammatory autoimmune heart disease.
A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.
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.
We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7µm. Using volume rendering software, this allows the reconstruction of internal structures in their natural state without the artefacts produced by histological sectioning.
Classical fear conditioning paradigm was adapted for human participants in a fully immersive virtual reality setting. Using a discrimination paradigm, conditioned fear, cue and context memory retention, and extinction was measured with skin conductance response to dynamic virtual snakes and spiders (the conditioned stimuli) in two distinct virtual contexts.
Microvolume samples are quantified by a spectrophotometer system that uses natural surface tension to retain samples without the use of cuvettes or capillaries. The dynamic range of protein concentrations and speed by which they can be measured are greatly increased with this method.
We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.
A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.
1Department of Paediatrics, Division of Infectious and Immunological Diseases, Child and Family Research Institute, University of British Columbia, 2Department of Computer Science, University of British Columbia, 3Department of Psychology, University of British Columbia
Visual analytics (VA) is a new approach of analyzing data interactively. In this video, we discuss the data overload problem brought on by high-throughput biological experiments, and propose VA as a solution to such problem. The video demonstrates analysis within and between immunological datasets using a VA tool called Tableau.
Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.