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 Bioengineering
Video-rate Scanning Confocal Microscopy and Microendoscopy
1Program in Biophysics, Harvard University, 2Division of Health Sciences and Technology, Harvard-MIT, 3Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School
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.
JoVE General
Label-free in situ Imaging of Lignification in Plant Cell Walls
1Energy Biosciences Institute, University of California, Berkeley, 2Molecular Foundry, Lawrence Berkeley National Laboratory, 3Physical Biosciences Division, Lawrence Berkeley National Laboratory
A method based on confocal Raman microscopy is presented that affords label-free visualization of lignin in plant cell walls and comparison of lignification in different tissues, samples or species.
JoVE Bioengineering
High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging
1Department of Bioengineering, Rice University, 2Department of Molecular and Cellular Oncology, The Univeristy of Texas M. D. Anderson Cancer Center
In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.
JoVE General
4D Imaging of Protein Aggregation in Live Cells
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.
JoVE Neuroscience
Isolation and Culture of Human Fungiform Taste Papillae Cells
1Monell Chemical Senses Center, 2New York University College of Dentistry, 3AFB International
We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.
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 Immunology and Infection
Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis
1Division of Microbiology, Tulane National Primate Research Center, 2Division of Comparative Pathology, Tulane National Primate Research Center, 3Division of Veterinary Medicine, Tulane National Primate Research Center
Dermatitis herpetiformis (DH) is a chronic inflammatory condition characterized by an autoimmune reaction between IgA and epidermal transglutaminase (eTG). DH develops in a very small portion of gluten-sensitive and/or celiac patients. The results of this study indicate that DH can also develop in a rhesus monkey host with symptoms of idiopatic dermatitis.
JoVE General
Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis
1Bioengineering, University of Pittsburgh, 2Developmental Biology, University of Pittsburgh
Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.
JoVE Neuroscience
Multiphoton Microscopy of Cleared Mouse Brain Expressing YFP
1Department of Biomedical Engineering, Yale University, 2Department of Biomedical Engineering, Louisiana Tech University
Multiphoton microscopy of whole mouse organs is possible by optically clearing the organ before imaging, but not all protocols preserve the fluorescent signal of fluorescent proteins. Using an optical clearing method with ethanol-based dehydration and benzyl alcohol:benzyl benzoate clearing, we show high-resolution multiphoton images of whole mouse brain expressing YFP.
JoVE Neuroscience
Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain
The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.
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 General
Photoconversion of Purified Fluorescent Proteins and Dual-probe Optical Highlighting in Live Cells
Department of Molecular Physiology and Biophysics, Vanderbilt University
This protocol describes a general approach to perform photoconversion of fluorescent proteins on a confocal laser scanning microscope. We describe procedures for the photoconversion of puried protein samples, as well as for dual-probe optical highlighting in live cells with mOrange2 and Dronpa.
JoVE General
Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination
1Department of Biology, University of Washington, 2Howard Hughes Medical Institute, University of Washington, 3PRESTO, Japan Science and Technology Agency
We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.
JoVE General
Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation
Department of Cellular Biology and Anatomy, Medical College of Georgia
In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.
JoVE General
Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans
1Department of Biological Sciences, University of Alabama in Huntsville, 2NIDDK-National Institutes of Health
This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.
JoVE Bioengineering
Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography
1Department of Biomedical Engineering, Northwestern University, 2Department of Physics, Harbin Institute of Technology, 3Department of Ophthalmology, University of Southern California, 4Department of Ophthalmology, Northwestern University
Photoacoustic ophthalmology (PAOM), an optical-absorption-based imaging modality, provides the complementary evaluation of the retina to the currently available ophthalmic imaging technologies. We report the using of PAOM integrated with spectral-domain optical coherence tomography (SD-OCT) for simultaneous multimodal retinal imaging in rats.
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 Neuroscience
A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain
1Department of Physics, University of California, San Diego, 2Department of Engineering Science and Mechanics, Pennsylvania State University, 3Department of Neurosurgery, Pennsylvania State University, 4Section of Neurobiology, University of California, San Diego
We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.
JoVE Neuroscience
Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction
1Lehrstuhl für Biomolekulare Sensoren, Technische Universität München, 2Center for Integrated Protein Science (Munich) at the Institute of Neuroscience, Technische Universität München, 3TUM Institute for Advanced Study and German Center for Neurodegenerative Diseases, Technische Universität München, 4Munich Cluster for Systems Neurology (SyNergy), Technische Universität München
Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.
JoVE General
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
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 Neuroscience
Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology
1Department of Biology, University of Iowa, 2Molecular Targeting Technologies, Inc.
A combination of different techniques to maximize data collection from mouse tissue is presented.
JoVE General
Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)
1Institute for Clinical Neurobiology, University of Wuerzburg, 2Department of Synapses - Circuits - Plasticity, Max Planck Institute of Neurobiology, Martinsried, 3Walter Brendel Centre of Experimental Medicine, Ludwig-Maximilians University of Munich
Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.
JoVE General
Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT)
1Institut National de la Santé et de la Recherche Médicale, UMR 631, Parc scientifique de Luminy, 2Centre National de la Recherche Scientifique, UMR 6102, Parc scientifique de Luminy, 3Centre d'Immunologie de Marseille-Luminy, Aix-Marseille University, 4École Centrale Marseille, Technopôle de Château-Gombert, 5Institut Fresnel, Aix-Marseille University, 6Centre National de la Recherche Scientifique, UMR 6133, Aix-Marseille University
Multiple-Target Tracing is a homemade algorithm developed for tracking individually labeled molecules within the plasma membrane of living cells. Efficiently detecting, estimating and tracing molecules over time at high-density provide a user-friendly, comprehensive tool to investigate nanoscale membrane dynamics.
JoVE Clinical and Translational Medicine
Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease
1Department of Pharmacology, University of Toledo, College of Pharmacy and Pharmaceutical Sciences, 2Department of Emergency and Intensive Care, ProMedica Sponsored Research
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.
JoVE Clinical and Translational Medicine
Probe-based Confocal Laser Endomicroscopy of the Urinary Tract: The Technique
1Department of Urology, Stanford University School of Medicine, 2Veterans Affairs Palo Alto Health Care System
Probe-based confocal laser endomicroscopy enables real-time microscopy of the human urinary tract during cystoscopy, providing dynamic, intravital imaging of pathological states such as bladder cancer with cellular resolution. Endomicroscopy may augment the diagnostic accuracy of standard white light endoscopy and provide intraoperative image guidance to improve surgical resection.
JoVE General
Obtaining Hemocytes from the Hawaiian Bobtail Squid Euprymna scolopes and Observing their Adherence to Symbiotic and Non-Symbiotic Bacteria
Department of Molecular and Cell Biology, University of Connecticut
This video will demonstrate how to obtain hemocytes (blood cells) from the Hawaiian bobtail squid, Euprymna scolopes for use in cell biological and bacterial adhesion assays. Hemocytes will be stained with a fluorescent dye and exposed to GFP-labeled bacteria.
JoVE Bioengineering
Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)
1Department of Electrical and Computer Engineering, Boston University, 2Department of Biomedical Engineering, Boston University, 3Center for Advanced Genomics Technology, Boston University, 4Department of Medicine, Section of Infectious Diseases, Boston University School of Medicine, 5Department of Microbiology, Boston University School of Medicine, 6CNR (National Research Council), Istituto di Chimica del Riconoscimento Molecolare
Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.
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 Neuroscience
Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo
Department of Cell Biology and Human Anatomy, School of Medicine, University of California, Davis
We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.
JoVE Bioengineering
Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy
1Department of Thoracic and Cardiovascular Surgery and Inter-University Centre for Medical Technology Stuttgart-Tübingen (IZST), Eberhard Karls University, Tübingen, 2Department of Cell and Tissue Engineering, Fraunhofer Institute of Interfacial Engineering and Biotechnology (IGB) Stuttgart, Germany, 3Department for Medical Interfacial Engineering (IGVT), University of Stuttgart, Germany, 4Institute of Tissue Engineering and Regenerative Medicine, Julius-Maximillians University, Würzburg, Germany
Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.
JoVE Neuroscience
Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation
Neurobiology and Anatomy, University of Rochester
In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.
JoVE Neuroscience
High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium
1Neural Development Group, Division of Cell and Developmental Biology, College of Life Sciences, University of Dundee, Dundee, UK, 2Wellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee, UK
Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.
JoVE Immunology and Infection
Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human α-actinin by Confocal Imaging
Department of Cellular and Molecular Medicine, University of Bristol, UK
Neisseria meningitidis (Nm), a gram negative human-specific respiratory pathogen, can bind to human α-actinin. Here we present a protocol for visualisation of colocalisation of the bacterium with intracellular α-actinin after bacterial entry into human brain microvascular endothelial cells (HBMECs).
JoVE Application Notes
The Waters Xevo™ TQ-S Equipped With New StepWave™ and ScanWave™ Technology - ADVERTISEMENT
Pharmaceutical Business Operations, Waters Corporation
JoVE General
Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging
This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.
JoVE Chemistry
Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)
Proteomics and Metabolomics Facility, Colorado State University
Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. This article outlines a typical workflow utilized for non-targeted metabolite profiling of serum including sample organization and preparation, data acquisition, data analysis, quality control, and metabolite identification.
JoVE Neuroscience
Vibratome Sectioning for Enhanced Preservation of the Cytoarchitecture of the Mammalian Organ of Corti
Department of Pediatrics, Children’s Research Institute, Medical College of Wisconsin
A simple procedure of vibratome sectioning the organ of Corti, followed by immunohistochemistry and confocal microscopy is described. This procedure allows for improved preservation of the fine cytoarchitecture of the mammalian organ of Corti, and consequently allows for accurate quantification of cell types.
JoVE General
A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium
Biosciences, College of Life and Environmental Sciences, The University of Exeter
We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.
JoVE Neuroscience
Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging
1Division of Biomedical Sciences, University of California, Riverside, 2Department of Bioengineering, University of California, Riverside
We present a method of creating a thinned-skull cortical window (TSCW) in a mouse model for in vivo OCT imaging of the cerebral cortex.
JoVE Applied Physics
Terahertz Microfluidic Sensing Using a Parallel-plate Waveguide Sensor
Department of Electrical and Computer Engineering, Rice University
The procedure for implementing a refractive index sensor for terahertz frequencies based on a grooved parallel-plate waveguide geometry is described here. The method yields a measurement of the refractive index of a small volume of liquid through monitoring of the shift in the resonant frequency of the waveguide structure
JoVE Applied Physics
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
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.
JoVE Bioengineering
Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and and Induced Pluripotent Stem Cells (iPSCs)
1Department of Medicine (Hematology, Oncology, and Transplant), University of Minnesota, Minneapolis, 2Stem Cell Institute, University of Minnesota, Minneapolis
This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.
JoVE General
NanoDrop Microvolume Quantitation of Nucleic Acids
Thermo Scientific NanoDrop Products, Wilmington, Delaware
The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.
JoVE Clinical and Translational Medicine
Corneal Confocal Microscopy: A Novel Non-invasive Technique to Quantify Small Fibre Pathology in Peripheral Neuropathies
Division of Cardiovascular Medicine, University of Manchester
Corneal Confocal microscopy is a non-invasive clinical technique which may be used to quantify C fibre damage to diagnose and stratify patients with increasing neuropathic severity.
JoVE General
In vivo Imaging of Deep Cortical Layers using a Microprism
Department of Biomedical Engineering, Yale University
Right-angle microprisms inserted into the mouse neocortex allows for deep imaging of multiple cortical layers with a viewpoint typically found in slice. One-millimeter microprisms offer a wide field-of-view (~900 μm) and spatial resolutions sufficient to resolve dendritic spines. We demonstrate layer V neuronal imaging and neocortical vascular imaging using microprisms.
JoVE Immunology and Infection
Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy
1Division of Infectious Diseases, Department of Medicine, Immunology Institute, Mount Sinai School of Medicine, 2NSF Center for Biophotonics, University of California, Davis, 3Structural and Computational Biology Unit, European Molecular Biology Laboratory
This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.
JoVE General
Upright Imaging of Drosophila Embryos
1Department of Biology, Case Western Reserve University, 2Department of Genetics, Case Western Reserve University
Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.
JoVE General
Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans
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.
JoVE General
In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution
1Junior Research Group Synaptic Plasticity, Hertie Institute for Clinical Brain Research, University of Tübingen, 2Graduate School of Cellular and Molecular Neuroscience, University of Tübingen
This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.
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