JoVE Immunology and Infection
1Department of Immunology and Infectious Diseases, Montana State University, 2Department of Molecular Biology, Princeton University
Live cell imaging of alphaherpes virus infections enables analysis of the dynamic events of directed transport and intercellular spread. Here, we present methodologies that utilize recombinant viral strains expressing fluorescent fusion proteins to facilitate visualization of viral assemblies during infection of primary neurons.
Published August 16, 2013. Keywords: Virology, Infection, Immunology, Medicine, Molecular Biology, Cellular Biology, Microbiology, Genetics, Microscopy, Fluorescence, Neurobiology, Herpes virus, fluorescent protein, epifluorescent microscopy, neuronal culture, axon, virion, video microscopy, virus, live cell, imaging
1Max-Planck-Institute for Molecular Biomedicine and Institute of Cell Biology, 2Department of Internal Medicine, Yale Cardiovascular Research Center and Section of Cardiovascular Medicine
This protocol describes a method of live cell imaging using primary rat neonatal cardiomyocytes following lentiviral and adenoviral transduction using confocal spinning disk microscopy. This enables detailed observations of cellular processes in living cardiomyocytes.
Published June 24, 2014. Keywords: Cellular Biology, live cell imaging, cardiomyocyte, primary cell culture, adenovirus, lentivirus, confocal spinning disk microscopy
1Centre for Vascular Research, University of New South Wales, 2School of Medical Sciences, University of New South Wales
Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.
Published February 19, 2015. Keywords: Bioengineering, Cell adhesion, biosensor, live cell imaging, extracellular matrix, fibronectin, mechanobiology, cell signaling, redox signaling, oxidative stress, myeloperoxidase, endothelium
1Signalling Programme, The Babraham Institute, 2MRC Group, Cardiff School of Biosciences, Cardiff University
Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.
Published July 27, 2013. Keywords: Cellular Biology, Molecular Biology, Biochemistry, Phosphatidylinositols, Microscopy, Fluorescence, Video, Autophagy, Cell Biology, Autophagy, Omegasome, DFCP1, LC3, Live imaging, Time-lapse microscopy, cell, imaging
1Department of Pharmacology, Wayne State University, 2Barbara Ann Karmanos Cancer Institute, Wayne State University
We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.
Published February 17, 2012. Keywords: Medicine, Immunology, Breast, cancer, extracellular matrix, invasion, proteolysis, tumor microenvironment
1University of California, Davis
Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.
Published December 22, 2011. Keywords: Bioengineering, cell invasion, three-dimensional matrix, collagen gel, live-cell confocal imaging, FRAP, GFP, epithelial cyst
1Department of Radiation Oncology, Virginia Commonwealth University, 2Department of Biochemistry & Molecular Biology, Virginia Commonwealth University, 3Department of Anatomy & Neurobiology, Virginia Commonwealth University, 4Massey Cancer Center, Virginia Commonwealth University
This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.
Published September 28, 2012. Keywords: Genetics, Molecular Biology, Cellular Biology, Biochemistry, DNA, Double-strand breaks, DNA damage response, proteins, live cell imaging, 3D cell imaging, confocal microscopy
1Department of Molecular and Cellular Biology, University of Guelph
Live-cell imaging of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte cell cultures using the NucView 488 caspase-3 substrate. This technique is applicable for programmed cell death assays in real-time in a variety of cell types and tissues.
Published January 13, 2012. Keywords: Neuroscience, myelin basic protein, apoptosis, neuroprotection, caspase-3, live-cell imaging, glia, oligodendrocytes
JoVE Immunology and Infection
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.
Published July 28, 2011. Keywords: Immunology, Optical trapping, optical tweezers, T-cell, pathogen, live cell imaging, spinning disk confocal microscopy, Aspergillus fumigatus, Candida albicans, fungi
1Epigenetics and Progenitor Cells Keystone, Fox Chase Cancer Center
In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae
Published May 27, 2011. Keywords: Neuroscience, Live imaging, asymmetric cell division, Drosophila, pupa
1Division of Chemical Biology, New England Biolabs
SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again.
Published May 17, 2010. Keywords: Cellular Biology, fluorescence, labeling, imaging, SNAP-tag, tag, microscopy, AGT, surface, intracellular, fusion
1Department of Biomedical Engineering, University of Wisconsin-Madison, 2Materials Science Program, University of Wisconsin-Madison, 3Department of Neurological Surgery, University of Wisconsin-Madison, 4Carbone Comprehensive Cancer Center and Center for Stem Cell and Regenerative Medicine, University of Wisconsin-Madison
A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.
Published December 23, 2011. Keywords: Medicine, BTSC, Tumor, cancer stem cell, cell migration, microfluidics, Glioblastoma Multiforme, GBM, chemotaxis, amoeboid, mesenchymal, haptotaxis, PDMS
1Department of Cell Biology, Scripps Institute
Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).
Published August 5, 2009. Keywords: Cellular Biology, FSM, qFSM, speckle, actin, cytoskeleton, fluorescence, microscopy, microinjection
JoVE Immunology and Infection
1Molecular Genetics Group, Groningen Biomolecular Sciences and Biotechnology Institute, Centre for Synthetic Biology, University of Groningen
This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.
Published July 28, 2011. Keywords: Immunology, time-lapse fluorescence microscopy, single cell analysis, cell history, cell growth, development, promoter activity, protein localization, GFP, Bacillus subtilis, Streptococcus pneumoniae
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.
Published May 23, 2010. Keywords: Developmental Biology, epithelial cells, frogs, Xenopus laevis, epithelia, multicellular, high-resolution confocal microscopy, animal cap explant, embryos
1Department of Physics and Astronomy, University of Maine
We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers within single cells.
Published December 9, 2013. Keywords: Basic Protocol, Microscopy, Super-resolution imaging, Multicolor, single molecule, FPALM, Localization microscopy, fluorescent proteins
1W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, 2School of Life Sciences, Chinese University of Hong Kong, 3Center for Cell Dynamics, Department of Biological Chemistry, Johns Hopkins University School of Medicine
The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis.
Published February 16, 2015. Keywords: Cellular Biology, Anastasis, apoptosis, apoptotic bodies, caspase, cell death, cell shrinkage, cell suicide, cytochrome c, DNA damage, genetic alterations, mitochondrial outer membrane permeabilization (MOMP), programmed cell death, reversal of apoptosis
1National Heart, Lung, and Blood Institute, National Institutes of Health
This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.
Published July 7, 2014. Keywords: Neuroscience, live imaging, Drosophila, neuroblast, stem cell, asymmetric division, centrosome, brain, cell cycle, mitosis
1The ithree Institute, University of Technology, Sydney
Spatiotemporal information about dynamic proteins inside live cells is crucial for understanding biology. A type of super-resolution microscopy called fast 3D-structured illumination microscopy (f3D-SIM) reveals unique information about the cytokinetic Z ring in bacteria: both its bead-like appearance and the rapid dynamics of FtsZ within the ring.
Published September 29, 2014. Keywords: Molecular Biology, super-resolution microscopy, fluorescence microscopy, OMX, 3D-SIM, Blaze, cell division, bacteria, Bacillus subtilis, Staphylococcus aureus, FtsZ, Z ring constriction
1Department of Anatomy and Cell Biology, University of Iowa Carver College of Medicine
Stage-specific isolation of mid-to-late Drosophila follicles is useful for a variety of purposes. Such follicles develop in culture, which allows for genetic and/or pharmacologic manipulations to be coupled with in vitro development assays and live imaging. Additionally, follicles can be used for molecular studies, such as isolating mRNA and protein.
Published December 2, 2013. Keywords: Developmental Biology, Drosophila melanogaster, Organ Culture Techniques, Gene Expression Profiling, Microscopy, Confocal, Cell Biology, Genetic Research, Molecular Biology, Pharmacology, Drosophila, oogenesis, follicle, live-imaging, gene expression, development
1Heart Research Center Goettingen, 2Clinic of Cardiology & Pulmonology, University Medical Center Goettingen, 3German Center for Cardiovascular Research (DZHK) partner site Goettingen, 4BioMET, Center for Biomedical Engineering & Technology, University of Maryland School of Medicine
In cardiac myocytes, tubular membrane structures form intracellular networks. We describe optimized protocols for i) isolation of myocytes from mouse heart including quality control, ii) live cell staining for state-of-the-art fluorescence microscopy, and iii) direct image analysis to quantify the component complexity and the plasticity of intracellular membrane networks.
Published October 15, 2014. Keywords: Bioengineering, cardiac myocyte, atria, ventricle, heart, primary cell isolation, fluorescence microscopy, membrane tubule, transverse-axial tubule system, image analysis, image processing, T-tubule, collagenase
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.
Published May 7, 2013. Keywords: Cellular Biology, Neurobiology, Neuroscience, Molecular Biology, Biochemistry, Biomedical Engineering, Bioengineering, Virology, Medicine, Anatomy, Physiology, Surgery, Endoplasmic Reticulum, ER, Calcium Signaling, calcium store, calcium imaging, calcium indicator, metabotropic signaling, Ca2+, neurons, cells, mouse, animal model, cell culture, targeted esterase induced dye loading, imaging
1Department of Structural Biology, University of Pittsburgh School of Medicine, 2Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine
We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.
Published June 24, 2013. Keywords: Bioengineering, Molecular Biology, Structural Biology, Virology, Biophysics, Cellular Biology, Physiology, Medicine, Biomedical Engineering, Infection, Microbiology, Technology, Industry, Agriculture, Life Sciences (General), Correlative microscopy, CryoET, Cryo-electron tomography, Confocal live-cell imaging, Cryo-fluorescence light microscopy, HIV-1, capsid, HeLa cell, cell, virus, microscopy, imaging
1Center for Genetic Medicine Research, Children's National Medical Center, 2Department of Integrative Systems Biology, George Washington University
The process of healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of cell membrane injury. This protocol describes assays to monitor these processes.
Published March 24, 2014. Keywords: Biochemistry, cell injury, lysosome exocytosis, repair, calcium, imaging, total internal reflection fluorescence (TIRF) microscopy, laser ablation
JoVE Immunology and Infection
1Research Group Phagosome Biology, Helmholtz Centre for Infection Research, 2Division of Mycobacterial Research, National Institute for Medical Research
Published March 10, 2014. Keywords: Immunology, Lysosome, Phagosome, phagolysosome, live-cell imaging, phagocytes, macrophages
1Department of Bioengineering, University of Pennsylvania, 2Integrated DNA Technologies, Inc.
Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.
Published August 6, 2014. Keywords: Genetics, RNA, imaging, single molecule, fluorescence, living cell
1Department of Otolaryngology, University of California, San Francisco, 2Department of Chemistry, University of California, Berkeley, 3Materials Science Division, Lawrence Berkeley National Laboratory, 4Department of Pharmaceutical Chemistry, University of California, San Francisco, 5Tetrad Graduate Program, University of California, San Francisco, 6Center for Systems and Synthetic Biology, University of California, San Francisco, 7Chemistry and Chemical Biology Graduate Program, University of California, San Francisco
We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.
Published October 23, 2014. Keywords: Bioengineering, monovalent quantum dots, single particle tracking, SNAP tag, steric exclusion, phosphorothioate, DNA, nanoparticle bioconjugation, single molecule imaging
1Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, 2San Raffaele Scientific Institute and Vita-Salute University, 3CEND Center of Excellence in Neurodegenerative Diseases, Università degli Studi di Milano
This paper provides a method for investigating neurotransmitter vesicle dynamics in neuroblastoma cells, using a synaptobrevin2-pHluorin construct and Total Internal Reflection Fluorescence Microscopy. The strategy developed for image processing and data analysis is also reported.
Published January 29, 2015. Keywords: Neuroscience, Synaptic vesicles, neurotransmission, Total Internal Reflection Fluorescence Microscopy, pHluorin, neuroblastoma cells
1Department of Horticulture and Landscape Architecture, Purdue University, 2Bindley Bioscience Center, Purdue University, 3Institute of Biotechnology, Jiangsu Academy of Agricultural Sciences, 4College of Environmental & Resource Science, Zhejiang University, 5Dryland Agriculture Research Centre, Shanxi Academy of Agricultural Sciences, 6Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences
Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.
Published September 2, 2014. Keywords: Plant Biology, Aequorin, Case12, abiotic stress, heavy metal stress, copper ion, calcium imaging, Arabidopsis
1Department of Molecular Physiology & Biophysics, University of Iowa Carver College of Medicine, 2Department of Biology & Biochemistry, University of Bath
We describe the use of styryl FM dyes to image synaptic vesicle recycling in functional nerve terminals. This protocol can be applied not only to evoked, but also spontaneous and miniature synaptic activities. The protocol expands the variety of synaptic events that can be effectively evaluated.
Published March 31, 2014. Keywords: Neuroscience, Presynaptic Terminals, Synaptic Vesicles, Microscopy, Biological Assay, Nervous System, Endocytosis, exocytosis, fluorescence imaging, FM dye, neuron, photobleaching
1Department of Cell Biology and Physiology, Washington University School of Medicine
Four-dimensional (4D) imaging is utilized to study the behavior and interactions among two types of endosomes in living vertebrate nerve terminals. Movement of these small structures is characterized in three dimensions, permitting confirmation of events such as endosome fusion and exocytosis.
Published April 16, 2014. Keywords: Neuroscience, Microscopy, Fluorescence, Endocytosis, nerve, endosome, lysosome, deconvolution, 3D, 4D, epifluorescence
1Department of Cell Biology and Molecular Genetics, University of Maryland
Live imaging of cells exposed to the lipophilic dye FM1-43 allows precise determination of the kinetics by which pore-forming toxins are removed from the plasma membrane. This is a sensitive assay that can be used to assess requirements for Ca2+, sphingomyelinase and other factors on plasma membrane repair.
Published August 25, 2013. Keywords: Cellular Biology, Molecular Biology, Infection, Medicine, Immunology, Biomedical Engineering, Anatomy, Physiology, Biophysics, Genetics, Bacterial Toxins, Microscopy, Video, Endocytosis, Biology, Cell Biology, streptolysin O, plasma membrane repair, ceramide, endocytosis, Ca2+, wounds
JoVE Developmental Biology
1Biology Department, Wesleyan University
This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.
Published January 12, 2015. Keywords: Developmental Biology, Drosophila melanogaster, pupal eye, pattern formation, ommatidial development, live cell imaging, motion stabilization, fluorescence microscopy
JoVE Immunology and Infection
1Microbiology Unit, Department of Biochemistry, University of Oxford, 2Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford
Photoactivated localization microscopy (PALM) combined with single-molecule tracking allows direct observation and quantification of protein-DNA interactions in live Escherichia coli cells.
Published March 10, 2014. Keywords: Immunology, Super-resolution microscopy, single-particle tracking, Live-cell imaging, DNA-binding proteins, DNA repair, molecular diffusion
1Department of Physiology, Louisiana State University Health Sciences Center
Microscopic imaging of live endothelial cells expressing GFP-actin allows characterization of dynamic changes in cytoskeletal structures. Unlike techniques that use fixed specimens, this method provides a detailed assessment of temporal changes in the actin cytoskeleton in the same cells before, during, and after various physical, pharmacological, or inflammatory stimuli.
Published November 18, 2011. Keywords: Cell Biology, Endothelial cells, actin, cytoskeleton, live-cell imaging, GFP, lamellipodia, stress fibers, kymograph analysis
JoVE Immunology and Infection
1The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University
We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.
Published June 23, 2013. Keywords: Immunology, Cellular Biology, Molecular Biology, Medicine, T-cell activation, Live-cell imaging, Signal transduction, Confocal microscopy, Signaling complex, Co-localization analysis, fluorescence, cell biology, T-cell, cell, imaging
1Department of Physiology and Neurobiology, University of Connecticut, 2Stem Cell Institute, University of Connecticut, 3Department of Neurology, Yale University School of Medicine
A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.
Published August 25, 2014. Keywords: Neuroscience, NG2, CSPG4, polydendrocyte, oligodendrocyte progenitor cell, oligodendrocyte, myelin, organotypic slice culture, time-lapse
1Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine
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.
Published January 21, 2013. Keywords: Biophysics, Cellular Biology, Microbiology, Molecular Biology, Structural Biology, Chemistry, Physics, super-resolution imaging, PALM, FtsZ, mEos2, cell division, cytokinesis, divisome
1Institute for Physiology, Pathophysiology, & Toxicology, Centre for Biomedical Research and Training (ZBAF), University of Witten/Herdecke, 2Institute for Immunology & Experimental Oncology, Centre for Biomedical Research and Training (ZBAF), University of Witten/Herdecke
Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.
Published October 28, 2014. Keywords: Cellular Biology, Kidney, FACS, second messenger, proximal tubule, calcium indicators, probenecid, endoplasmic reticulum, ionomycin
1Emmy Noether Group of the DFG, Department of Cardiology and Pneumology, European Heart Research Insitute Göttingen, Georg August University Medical Center, Göttingen, Germany
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.
Published August 20, 2012. Keywords: Molecular Biology, Medicine, Cellular Biology, FRET, microscope, imaging, software, cAMP, biosensor
1German Center for Neurodegenerative Diseases, DZNE, 2Laboratory of Functional Neurogenomics, Department of Neurodegenerative Diseases, Hertie Institute for Clinical Brain Research, University of Tübingen
Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.
Published October 3, 2012. Keywords: Medicine, Genetics, Cellular Biology, Physiology, Parkinson's disease, fibroblasts, mitochondria, live cell imaging, mitochondrial function, mitochondrial morphology, mitophagy
1MRC Human Genetics Unit, MRC IGMM, Western General Hospital, University of Edinburgh
We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.
Published May 19, 2014. Keywords: Developmental Biology, mouse, melanoblast, skin, confocal microscopy, air-liquid interface
1Center for Neuroscience, Swammerdam Institute for Life Sciences, University of Amsterdam, 2Van Leeuwenhoek Centre for Advanced Microscopy, Section Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam
This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.
Published May 4, 2014. Keywords: Neuroscience, Dendritic Spine, Microscopy, Confocal, Fluorescence, Neurosciences, hippocampus, primary neuron, super resolution microscopy, structured illumination microscopy (SIM), neuroscience, dendrite
1Biological Sciences Platform, Sunnybrook Research Institute, 2Department of Otolaryngology - Head and Neck Surgery, University of Toronto, 3Department of Laboratory Medicine and Pathobiology, University of Toronto
Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.
Published November 2, 2014. Keywords: Bioengineering, Live-imaging, time lapse, cochlea, ear, reporter mouse, development, incubator microscope, Sox2
1Institute for Biomaterials and Biomedical Engineering, University of Toronto, 2Lyndhurst Centre, Toronto Rehabilitation Institute, 3Department of Surgery, University of Toronto
In this protocol we demonstrate how to construct custom chambers that permit the application of a direct current electric field to enable time-lapse imaging of adult brain derived neural precursor cell translocation during galvanotaxis.
Published October 13, 2012. Keywords: Neuroscience, Biomedical Engineering, Cellular Biology, Physiology, Molecular Biology, neural precursor cells, galvanotaxis, cell migration, time-lapse imaging, electric fields
JoVE Immunology and Infection
1Abteilung Mikrobiologie, Fachbereich Biologie/Chemie, Universität Osnabrück
This article describes methods for the synthesis and fluorescent labeling of nanoparticles (NPs). The NPs were applied in pulse-chase experiments to label the endo-lysosomal system of eukaryotic cells. Manipulation of the endo-lysosomal system by activities of the intracellular pathogen Salmonella enterica were followed by live cell imaging and quantified.
Published January 2, 2015. Keywords: Immunology, fluorescent nanoparticles, endo-lysosomal system, labeling, intracellular bacteria, quantitative image analysis, tubular compartments
1Department of Chemical and Biomolecular Engineering, Russ College of Engineering and Technology, Ohio University, 2Biomedical Engineering Program, Ohio University
Dual camera emission splitting systems for two-color fluorescence microscopy generate real-time image sequences with exceptional optical and temporal resolution, a requirement of certain live cell assays including parallel plate flow chamber adhesion assays. When software is employed to merge images from simultaneously acquired emission channels, pseudocolored image sequences are produced.
Published September 4, 2013. Keywords: Bioengineering, Cellular Biology, Biomedical Engineering, Molecular Biology, Biophysics, Biochemistry, Chemical Engineering, Chemistry, Cell Adhesion Molecules, Biological Markers, Antigens, Cell Adhesion Molecules, Microscopy, Fluorescence, Cell Physiological Processes, Cell Adhesion, Cell Physiological Phenomena, Colocalization, cell rolling, two-color fluorescence, cell, imaging
1Department of Oncology, Georgetown University, 2Lombardi Comprehensive Cancer Center, Georgetown University, 3Stem Cell Dynamics, Helmholtz Zentrum München - German Research Center for Environmental Health, 4Department of Medicine, Georgetown University, 5Department of Nanobiomedical Science and WCU Research Center of Nanobiomedical Science, Dankook University
Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.
Published February 8, 2013. Keywords: Cancer Biology, Medicine, Cellular Biology, Molecular Biology, Anatomy, Physiology, Oncology, Mammary Glands, Animal, Epithelial Cells, Mice, Genetically Modified, Primary Cell Culture, Time-Lapse Imaging, Early Detection of Cancer, Models, Genetic, primary cell culture, preneoplastic mammary epithelial cells, genetically engineered mice, time-lapse imaging, BRCA1, animal model
1Department of Biological Sciences, Carnegie Mellon University
Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.
Published October 20, 2014. Keywords: Cellular Biology, Endocytosis, TIRF, total internal reflection fluorescence microscopy, clathrin, arrestin, receptors, live-cell microscopy, clathrin-mediated endocytosis
1Fondazione Filarete, 2Department of Biotechnology and Translational Medicine, University of Milan, 3Neuroscience Institute, National Research Council (CNR), 4Department of Health Science, "Magna Graecia" University of Catanzaro
We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.
Published February 18, 2014. Keywords: Microbiology, Endoplasmic reticulum (ER), fluorescent proteins (FPs), confocal microscopy, fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP), ultrastructure, transmission electron microscopy (TEM)