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.
1Chemical and Biomolecular Engineering Department, University of Houston
Confocal microscopy is used to image quiescent and flowing colloid-polymer mixtures, which are studied as model systems for attractive suspensions. Image analysis algorithms are used to calculate structural and dynamic metrics for the colloidal particles that measure changes due to geometric confinement.
Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy
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.
The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy
1Physiology and Bosch Institute, University of Sydney, 2Motor Neuron Disease Research Group, Australian School of Advanced Medicine, Macquarie University, 3Advanced Microscopy Facility, Bosch Institute, University of Sydney
The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.
Published December 26, 2014. Keywords: Neuroscience, neuromuscular, motor endplate, motor control, sarcopenia, myasthenia gravis, amyotrophic lateral sclerosis, morphometry, confocal, immunofluorescence
1Cancer Research Center, Sheba Medical Center, 2The Neufeld Cardiac Research Institute, Tel-Aviv University, 3Department of Physiology and Pharmacology, Tel-Aviv University, 4Imaging Unit, Sackler Faculty of Medicine, Tel-Aviv University, 5Biotechnology and Cell Signaling, Ecole Superieure de Biotechnologie Strasbourg, 6Department of Human Molecular Genetics and Biochemistry, Tel-Aviv University
The cytotoxic activity of the phenanthridine PJ-34 in cancer cells undergoing mitosis was documented in real time by live confocal imaging. PJ-34 eradicated human breast cancer MDA-MB-231 cells harboring extra-centrosomes in mitosis. Unlike normal bi-focal mitosis, the extra-centrosomes were not clustered in the two spindle poles in the presence of PJ-34.
Published August 21, 2013. Keywords: Cancer Biology, Medicine, Cellular Biology, Molecular Biology, Biomedical Engineering, Anatomy, Physiology, Genetics, Neoplastic Processes, Pharmacologic Actions, Live confocal imaging, Extra-centrosomes clustering/de-clustering, Mitotic Catastrophe cell death, PJ-34, myocardial infarction, microscopy, imaging
In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy
1Light Microscopy Core Facility, NHLBI/NIH, 2Hematology Branch, NHLBI/NIH
Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.
Published August 6, 2014. Keywords: Stem Cell Biology, LeGO imaging, clonal tracking, fluorescent proteins, confocal microscopy, multiphoton microscopy, hematopoiesis, lentiviral vectors, hematopoietic stem cells
Confocal Time Lapse Imaging as an Efficient Method for the Cytocompatibility Evaluation of Dental Composites
1Laboratoire des Multimatériaux et Interfaces, UMR CNRS 5615, Université Lyon1, 2UFR d'Odontologie, Université Lyon1; Service de Consultations et de Traitements Dentaires, Hospices Civils de Lyon, 3UFR d'Odontologie, Université Paris Diderot; Service d'Odontologie, APHP, Hôpital Rothschild
The most studied aspect of the biocompatibility of dental composites is their cytotoxicity 3D CLSM time lapse imaging combined with fluorescent signal quantification allows sensitive evaluation of the cell fate over time. Utilizing this method could be an efficient tool to assess the cytocompatibility of dental composites.
1Mitochondria and Metabolism Center, Department of Anesthesiology and Pain Medicine, University of Washington
Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.
Published November 5, 2013. Keywords: Physiology, Heart Diseases, Metabolic Diseases, Microscopy, Confocal, Time-Lapse Imaging, Physiological Processes, Confocal imaging, mt-cpYFP transgenic mice, Superoxide flashes, Single mitochondrial measurement, Langendorff perfused heart, Skeletal muscles, in vivo
Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury
1Department of Neuroscience, IRCCS - Istituto di Ricerche Farmacologiche Mario Negri
A way to gain new insights into the complexity of the brain inflammatory response is presented. We describe immunofluorescence-based protocols followed by three-dimensional confocal analysis to investigate the pattern of co-expression of microglia/macrophage phenotype markers in a mouse model of focal ischemia.
Published September 4, 2013. Keywords: Neurobiology, Neuroscience, Molecular Biology, Cellular Biology, Medicine, Biomedical Engineering, Anatomy, Physiology, Central Nervous System Diseases, Neurodegenerative Diseases, biology (general), immunology, life sciences, animal models, Inflammation, stroke, alternative activation, brain injury, brain, imaging, confocal microscopy, three-dimensional imaging, clinical techniques, mouse, animal model
1Institute for Cell and Molecular Biology, The University of Texas at Austin
Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.
Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy
1Department of Oncology, INSERM U661, Functional Genomic Institute, 2Department of Anatomy, University of California
By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.
Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy
1Department of Pediatrics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, 2Biophotonics Core Facility, Centre de Recherche Clinique Etienne-Le Bel
Several methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of cytoplasmic and organellar pH. Here, we describe in detail a rapid and accurate methodology to simultaneously measure cytoplasmic and vesicular pH by ratiometric imaging of living cells.
1Department of Medicine, Roswell Park Cancer Institute, 2Department of Medicine, School of Medicine and Biomedical Sciences, University of Buffalo
A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.
Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration
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.
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.
Published January 10, 2013. Keywords: Medicine, Anatomy, Physiology, Cancer Biology, Surgery, Basic Protocols, Confocal laser endomicroscopy, microscopy, endoscopy, cystoscopy, human bladder, bladder cancer, urology, minimally invasive, cellular imaging
Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy
1European Laboratory for Non-linear Spectroscopy (LENS), 2Integrated Research Centre, University Campus Bio-medico of Rome, 3DAEMI, University of Cassino, 4National Institute of Optics (CNR-INO), 5Allen Institute for Brain Science, 6Department of Physics, University of Florence, 7ICON Foundation, Sesto Fiorentino, Italy
In this article we describe the full experimental procedure to reconstruct, with high resolution, the fine brain anatomy of fluorescently labeled mouse brains. The described protocol includes sample preparation and clearing, specimen mounting for imaging, data post-processing and multi-scale visualization.
Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human α-actinin by Confocal Imaging
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).
1School of Biological Sciences, Monash University, 2Janelia Farm Research Campus, Howard Hughes Medical Institute
Time-lapse microscopy allows the visualization of developmental processes. Growth or drift of samples during image acquisition reduces the ability to accurately follow and measure cell movements during development. We describe the use of open source image processing software to correct for three dimensional sample drift over time.
Published April 12, 2014. Keywords: Bioengineering, Image Processing, Computer-Assisted, Zebrafish, Microscopy, Confocal, Time-Lapse Imaging, imaging, zebrafish, Confocal, fiji, three-dimensional, four-dimensional, registration
Corneal Confocal Microscopy: A Novel Non-invasive Technique to Quantify Small Fibre Pathology in Peripheral Neuropathies
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.
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.
1Université Paris Descartes Sorbonne Paris Cité, INSERM UMR-S970, 2Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Service de Radiologie
In this paper, we present a method to analyze tumor microvessels in vivo using dynamic contrast-enhanced fluorescence videomicroscopy. Two quantitative parameters were acquired: functional capillary density reflecting the vascularity of the tumor, and index leakage reflecting the leakiness of the endothelial wall.
Published September 11, 2013. Keywords: Medicine, Cancer, Biological, Microcirculation, optical imaging devices (design and techniques), Confocal videomicroscopy, microcirculation, capillary leakage, FITC-Dextran, angiogenesis
A Faster, High Resolution, mtPA-GFP-based Mitochondrial Fusion Assay Acquiring Kinetic Data of Multiple Cells in Parallel Using Confocal Microscopy
1Department of Neuroscience, Center for Neuroscience Research, Tufts School of Medicine, 2Department of Internal Medicine, Geriatrics & Gerontology, Wake Forest Baptist Medical Center, 3Department of Medicine, Boston University Medical Center
Mitochondrial fusion was measured by tracking the equilibration of photoconverted matrix-targeted GFP across the mitochondrial network over time. Thus far, only one cell could be subjected to an hour long kinetic analysis at a time. We present a method that simultaneously measures multiple cells, thereby speeding up the data collection process.
Published July 20, 2012. Keywords: Molecular Biology, Genetics, Cellular Biology, Physics, confocal microscopy, mitochondria, fusion, TMRE, mtPAGFP, INS1, mitochondrial dynamics, mitochondrial morphology, mitochondrial network
1Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), 2Inserm, U1016, Paris, France
This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.
Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.
Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy
1Department of Orthodontics and Maxillofacial Orthopedics, Medical University of Graz, 2Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, 3Department of Prosthodontics, Restorative Dentistry, Periodontology and Implantology, Medical University of Graz, 4Institute of Plant Sciences, Karl-Franzens-University Graz
We present a protocol for structural and compositional analysis of natural oral biofilm from orthodontic appliances with in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Oral biofilm samples were collected from palatal expanders, scraping acrylic-resin flakes off their surface and referring them for molecular processing.
Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy
Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.
Published September 30, 2013. Keywords: Developmental Biology, Craniofacial Abnormalities, Jaw Abnormalities, Cleft Palate, Craniofacial Abnormalities, Maxillofacial Abnormalities, Reconstructive Surgical Procedures, Developmental Biology, Embryology, Congenital, Hereditary, and Neonatal Diseases and Abnormalities, Craniofacial development, cranial neural crest, confocal microscopy, fate mapping, cell lineage analysis, sox10, kaede, photoconversion, zebrafish, palate
Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination
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.
Published December 31, 2012. Keywords: Plant Biology, Molecular Biology, Developmental Biology, Cellular Biology, Botany, plant, live imaging, epidermis, stomata, confocal, time lapse, Arabidopsis, cotyledon
In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.
Published April 1, 2009. Keywords: Neuroscience, Developmental Biology, brain development, zebrafish, morphogenesis, microinjection, single cell injection, live imaging, confocal microscopy, embryo mounting
Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.
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
A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening
1Inserm U1019 - CNRS UMR 8024, Institut Pasteur de Lille, Université de Lille
Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host cell using automated confocal microscopy.
Published January 17, 2014. Keywords: Infection, Mycobacterium tuberculosis, High-content/High-throughput screening, chemogenomics, Drug Discovery, siRNA library, automated confocal microscopy, image-based analysis
1School of Molecular Medical Sciences, University of Nottingham, 2Division of Drug Delivery and Tissue Engineering, University of Nottingham, 3Laboratory of Biophysics and Surface Analysis, University of Nottingham
Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.
1Biological Sciences Division, Pacific Northwest National Laboratory, 2Environmental Molecular Science Laboratory, Pacific Northwest National Laboratory, 3Structural Proteomics Group, Ontario Center for Structural Proteomics, University of Toronto, 4Center for Bioproducts and Bioenergy, Washington State University
Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.
1Department of Nanomedicine, Houston Methodist Research Institute, 2MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry and Chemical Engineering, Sun Yat-sen University, 3Pediatrics Department of Union Hospital, Huazhong University of Science and Technology, 4CAS Key Laboratory for Biomedical Effects of Nanomaterials & Nanosafety, National Center for Nanoscience & Technology of China, 5Department of Medicine, Weill Cornell Medical College, 6Department of Cell and Developmental Biology, Weill Cornell Medical College
Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.
1Department of Biology, Concordia University
This protocol describes how to image dividing cells within a tissue in Caenorhabditis elegans embryos. While several protocols describe how to image cell division in the early embryo, this protocol describes how to image cell division within a developing tissue during mid late embryogenesis.
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
1Section of Microbiology, MRC Centre for Molecular Bacteriology and Infection, Imperial College London, 2Département de Biologie du Développement et des Cellules Souches, Institut Pasteur, Unité Macrophages et Développement de l'Immunité
To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.
An injury paradigm using the Drosophila larval ventral nerve cord to investigate central nervous system regeneration and repair is described. Stabbing followed by laser scanning confocal microscopy in time-lapse and fixed specimens, combined with quantitative analysis with purposefully developed software and genetics, are used to investigate the molecular mechanisms of CNS regeneration and repair.
Published March 28, 2013. Keywords: Neurobiology, Developmental Biology, Neuroscience, Molecular Biology, Cellular Biology, Anatomy, Physiology, Bioengineering, Central Nervous System, Neuroglia, Drosophila, fruit fly, animal models, Wounds and Injuries, Cell Physiological Phenomena, Genetic Phenomena, injury, repair, regeneration, central nervous system, ventral nerve cord, larva, live imaging, cell counting, Repo, GS2, glia, neurons, nerves, CNS, animal model
Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development
1Cell and Developmental Biology, University of Michigan, 2Department of Biosciences and Nutrition, Karolinska Instituet Novum
Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.
Published September 4, 2014. Keywords: Molecular Biology, Developmental Biology, morphogenesis, mouse fetal intestine, whole organ culture, live imaging, cell signaling, three-dimensional reconstruction, two-photon imaging
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.
Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy
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
1Synthetic Biology Platform, Wyss Institute for Biologically Inspired Engineering, Harvard University, 2Department of Systems Biology, Harvard Medical School, 3Department of Biotechnology, Delft University of Technology
This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.
Published April 18, 2014. Keywords: Environmental Sciences, Plant Leaves, Synthetic Biology, Plants, Genetically Modified, DNA, Plant, RNA, Gene Targeting, Plant Physiological Processes, Genes, Gene gun, Gibson assembly, Nicotiana benthamiana, Alternative splicing, confocal microscopy, chloroplast, peroxisome
1Department of Molecular, Cellular and Developmental Biology, University of Michigan, 2Department of Biomedical Engineering, University of Michigan, 3Life Sciences Institute, University of Michigan, 4Department of Cell and Developmental Biology, University of Michigan, 5Department of Mechanical Engineering, University of Michigan
Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the 'larva chip' for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.
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
1Department of Biomedical Engineering, University of Southern California, 2Department of Biomedical Sciences, Cedars-Sinai Medical Center, 3Geffen School of Medicine, University of California, Los Angeles
This article details the procedures for optical imaging analysis of the tumor-targeted nanoparticle, HerDox. In particular, detailed use of the multimode imaging device for detecting tumor targeting and assessing tumor penetration is described here.
Published June 18, 2013. Keywords: Biomedical Engineering, Cancer Biology, Medicine, Bioengineering, Molecular Biology, Cellular Biology, Biochemistry, Nanotechnology, Nanomedicine, Drug Delivery Systems, Molecular Imaging, optical imaging devices (design and techniques), HerDox, Nanoparticle, Tumor, Targeting, Self-Assembly, Doxorubicin, Human Epidermal Growth Factor, HER, HER2+, Receptor, mice, animal model, tumors, imaging
Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion (STED) Nanoscopy
1Center for Human Immunobiology, Texas Children's Hospital and Baylor College of Medicine
Here we illustrate the protocol for imaging by two-color STED nanoscopy the cytotoxic immune synapse of NK cells recapitulated on glass. Using this method we obtain sub-100 nm resolution of synapse proteins and the cytoskeleton.
1Department of Biology, University of Texas at Tyler, 2U. S. Horticultural Research Laboratory, USDA ARS
Here we present a protocol to propagate Homalodisca vitripennis cells and HoCV-1 in vitro. Medium was removed from HoCV-1 positive cultures and RNA extracted every 24 hr for 168 hr. Cell survivability was quantified by trypan blue staining. Whole virus particles were extracted post-infection. Extracted RNA was quantified by qRT-PCR.
Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo
We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.
Published May 20, 2011. Keywords: Developmental Biology, Confocal microscopy, whole-mount immunohistochemistry, mouse embryo, blood vessel, lymphatic vessel, vascular patterning, arterial differentiation
A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy
1Department of Mechanical Science and Engineering, University of Illinois at Urbana-Champaign
Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 µm from the gel surface.
Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies
1Institut Curie, Centre de Recherche, CNRS, UMR 168, PhysicoChimie Curie, Université Pierre et Marie Curie, 2Kavli Institute for Brain and Mind, University of California, San Diego, 3Molecular Physiology and Biophysics Section, National Institute for Neurological Disorders and Stroke, National Institute of Health
The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods — electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.
Published January 22, 2015. Keywords: Biochemistry, Biomimetic model system, Giant Unilamellar Vesicle, reconstitution, ion channel, transmembrane protein, KvAP, electroformation, gel assisted swelling, agarose, inside-out patch clamp, electrophysiology, fluorescence microscopy