Contrast Enhanced Vessel Imaging using MicroCT
1Greehey Children's Cancer Research Institute, University of Texas Health Science Center at San Antonio, 2Small Animal Imaging Resources facility, University of Texas Health Science Center at San Antonio, 3Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, 4Department of Pediatrics, University of Texas Health Science Center at San Antonio
Contrast enhanced small animal vessel imaging by microCT is a rapid, cost-effective and high-throughput technique for serial in situ examination for tumor development, for analyzing the network of blood vessels that nourish them, and for following the response of tumors to preclinical therapeutic intervention(s).
Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells
1CYTOO Cell Architects, Grenoble, France, 2Centre Commun de Quantimétrie, Faculté de Médecine Rockefeller, Lyon, France
Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.
Single Particle Electron Microscopy Reconstruction of the Exosome Complex Using the Random Conical Tilt Method
Molecular Biophysics and Biochemistry, Yale University
This article describes a standard method to get a three-dimensional (3D) reconstruction of biological macromolecules using negative staining electron microscopy (EM). In this protocol, we explain how to get the 3D structure of the Saccharomyces cerevisiae exosome complex at medium resolution using the random conical tilt reconstruction method (RCT).
Zebrafish Brain Ventricle Injection
1Whitehead Institute for Biochemical Research, 2MIT - Massachusetts Institute of Technology
After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.
Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction
Department of Structural Biology, University of Pittsburgh School of Medicine
This article describes a method to obtain a three-dimensional (3D) structure of helically assembled molecules using cryo-electron microscopy. In this protocol, we use HIV-1 capsid assemblies to illustrate the detailed 3D reconstruction procedure for achieving a density map by the iterative helical real-space reconstruction method.
Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy
1Department of Biochemistry and Molecular Biology, LSU School of Medicine, 2Department of Oral Biology, LSU School of Dentistry, 3Stanley S. Scott Cancer Center, LSU School of Medicine
This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.
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.
Quantitative Visualization and Detection of Skin Cancer Using Dynamic Thermal Imaging
Department of Mechanical Engineering, The Johns Hopkins University
We demonstrated that malignant pigmented lesions with increased metabolic activity generate quantifiable amounts of heat and the measurement of the transient thermal response of the skin to a cooling excitation allows quantitative identification of melanoma and other skin cancers (vs. non-proliferative nevi) at an early stage of the disease.
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.
Time-lapse Imaging of Mitosis After siRNA Transfection
1Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah, 2Fluorescence Microscopy Core Facility, University of Utah
Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.
Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation
1Department of Pathology, Harvard Medical School, 2Massachusetts General Hospital, 3Wellman Center for Photomedicine, Harvard Medical School, 4Pulmonary and Critical Care Unit, Massachusetts General Hospital, 5Pulmonary and Critical Care Unit, Harvard Medical School
A method to image ex vivo pulmonary resection specimens with optical frequency domain imaging (OFDI) and obtain precise correlation to histology is described, which is essential to developing specific OFDI interpretation criteria for pulmonary pathology. This method is applicable to other tissue types and imaging techniques to obtain precise imaging to histology correlation for accurate image interpretation and assessment. Imaging criteria established with this technique would then be applicable to image assessment in future in vivo studies.
December 2011: This Month in JoVE
Here are some highlights from the December 2011 Issue of Journal of Visualized Experiments (JoVE).
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.
How to Measure Cortical Folding from MR Images: a Step-by-Step Tutorial to Compute Local Gyrification Index
1Department of Psychiatry, University of Geneva School of Medicine, 2Signal Processing Laboratory, École Polytechnique Fédérale de Lausanne, 3Department of Radiology, University Hospital Center and University of Lausanne, 4Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital
Measuring gyrification (cortical folding) at any age represents a window into early brain development. Hence, we previously developed an algorithm to measure local gyrification at thousands of points over the hemisphere1. In this paper, we detail the computation of this local gyrification index.
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.
Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain
1Department of Surgery, University of Texas Medical Branch, 2Department of Neuroscience and Cell Biology, University of Texas Medical Branch
A method for large-scale purification of the APP intracellular domain (AICD) is described. We also describe methodology to induce in vitro AICD aggregation and visualization by atomic force microscopy. The methods described are useful for biochemical/structural characterization of the AICD and the effects of molecular chaperones on its aggregation.
C. elegans Tracking and Behavioral Measurement
1Donnelly Centre, University of Toronto, 2Department of Physics and Astronomy, Vrije Universiteit, 3Okinawa Institute of Science and Technology, 4Department of Physics, University of Toronto
We have developed a video-rate tracking microscope system that can record and quantify C. elegans behavior at high resolution and high speeds. We have also developed computational methods to reduce the dimensionality of the worm images to a fundamental set of measurements that completely describe the shape of the worm.
Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells
1Department of Chemical and Biomolecular Engineering, University of Houston, 2Division of Pediatrics, Research Unit 907, University of Texas MD Anderson Cancer Center
We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.
Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone
1Electrical Engineering Department, University of California, Los Angeles, 2Bioengineering Department, University of California, Los Angeles, 3California NanoSystems Institute (CNSI), University of California, Los Angeles
We review our recent results on the integration of fluorescent microscopy and imaging flow cytometry tools on a cell-phone using compact and cost-effective opto-fluidic attachments. These cell-phone based micro-analysis devices might be useful for cytometric analysis, such as performing various cell counting tasks as well as for high-throughput screening of e.g., water samples in resource limited settings.
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.
C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays
1Department of Molecular Biology, Lewis-Sigler Institute for Integrative Genomics, Princeton University, 2Lewis-Sigler Institute for Integrative Genomics, Princeton University
Here we describe methods to test C. elegans associative learning and short- and long-term associative memory. These population assays employ the worms abilities to chemotax toward volatile odorants, and form positive associations upon pairing food with the chemoattractant butanone. Increasing the number of conditioning periods induces long-term memory.
Real-time fMRI Biofeedback Targeting the Orbitofrontal Cortex for Contamination Anxiety
1Department of Diagnostic Radiology, Yale University School of Medicine, 2Department of Psychiatry, Yale University School of Medicine, 3Yale Child Study Center, Yale University School of Medicine, 4Interdepartmental Neuroscience Program, Yale University School of Medicine
Here we present a method for training people to control a brain area involved in contamination anxiety and for probing the relationship between contamination anxiety and brain connectivity patterns.
Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy
1AG Cellular Dynamics and Cell Patterning, Max Planck Institute of Biochemistry, 2Helmholtz Zentrum München
Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.
Lensless On-chip Imaging of Cells Provides a New Tool for High-throughput Cell-Biology and Medical Diagnostics
1Electrical Engineering Department, University of California, Los Angeles, 2California NanoSystems Institute, University of California, Los Angeles
Lensfree on-chip imaging and characterization of cells is illustrated. This on-chip cell imaging approach provides a compact and cost-effective tool for medical diagnostics and high-throughput cell biology applications, making it especially suitable for resource poor settings.
Measuring the Induced Membrane Voltage with Di-8-ANEPPS
Department of Biomedical Engineering, Faculty of Electrical Engineering, University of Ljubljana
External electric field induces a voltage on the membrane of a cell, termed the induced membrane voltage (ΔΦ). By using the potentiometric dye di-8-ANEPPS, it is possible to measure the ΔΦ noninvasively. This video shows the protocol for measuring ΔΦ using di-8-ANEPPS.
Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor
Department of Neuroscience and Cell Biology, University of Texas Medical Branch
This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.
Glycan Profiling of Plant Cell Wall Polymers using Microarrays
1Australian Centre of Excellence in Plant Cell Walls, School of Botany, University of Melbourne, 2Plant Cell Biology Research Centre, School of Botany, University of Melbourne, 3CSIRO Plant Industry, Black Mountain Laboratories, 4Department of Plant Biology and Biotechnology, University of Copenhagen
A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.
Automated Midline Shift and Intracranial Pressure Estimation based on Brain CT Images
1Department of Biostatistics, Virginia Commonwealth University, 2Virginia Commonwealth University Reanimation Engineering Science (VCURES) Center, 3Department of Computer Science, Virginia Commonwealth University, 4Department of Radiology, Virginia Commonwealth University, 5Department of Emergency Medicine, Virginia Commonwealth University
An automated midline shift estimation and intracranial pressure (ICP) pre-screening system based on computed tomography (CT) images for patients with traumatic brain injury (TBI) is proposed using image processing and machine learning techniques.
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.
Imaging Odor-Evoked Activities in the Mouse Olfactory Bulb using Optical Reflectance and Autofluorescence Signals
This article presents the protocols of intrinsic optical signals and flavoproteins autofluorescence signals imaging to map odor-evoked activities at the surface of the olfactory bulb in mice.
Customization of Aspergillus niger Morphology Through Addition of Talc Micro Particles
Institute of Biochemical Engineering, Technische Universität Braunschweig
A method to precisely generate and to comprehensively characterize morphology of filamentous fungus Aspergillus niger is described, which allows the mathematical correlation of morphological appearance and productivity.
Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish
1Institute for Toxicology and Genetics, Karlsruhe Institute of Technology, Karlsruhe, Germany, 2Institute for Applied Informatics, Karlsruhe Institute of Technology, Karlsruhe, Germany
Here we describe a novel high-content chemically induced inflammation assay aiming at the identification of immune-modulatory bioactives. We have successfully combined automated microscopy with custom developed software scripts enabling automated quantification of the inflammatory response as well as further data processing, analysis, mining, and storage.
3D Printing of Preclinical X-ray Computed Tomographic Data Sets
1Department of Chemistry and Biochemistry, University of Notre Dame, 2Freimann Life Science Center, University of Notre Dame, 3Department of Biological Sciences, University of Notre Dame, 4Notre Dame Integrated Imaging Facility, University of Notre Dame, 5MakerBot Industries LLC, 6Departments of Biological Sciences, Aerospace and Mechanical Engineering, and Anthropology, University of Notre Dame, 7Harper Cancer Research Institute, University of Notre Dame
Using modern plastic extrusion and printing technologies, it is now possible to quickly and inexpensively produce physical models of X-ray CT data taken in a laboratory. The three -dimensional printing of tomographic data is a powerful visualization, research, and educational tool that may now be accessed by the preclinical imaging community.
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.
Live Imaging of GFP-labeled Proteins in Drosophila Oocytes
Department of Biology, Vassar College
A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.
In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee
1Institut für Biologie - Neurobiologie, Freie Universität Berlin, 2Institut für Biologie - Neurobiologie, Free University Berlin - Freie Universitaet Berlin
Bees can be conditioned in an appetitive olfactory learning paradigm (PER-conditioning). Using odors as stimuli, we established a method in which behavior is recorded while simultaneously Calcium Imaging is used to measure odor evoked activity in mushroom body neurons in vivo.
In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis
1Cardiovascular Research Center and Cardiology Division, Massachusetts General Hospital, Harvard Medical School, 2Institute for Biological and Medical Imaging, Helmholtz Zentrum München und Technische Universität München, 3Department of Electrical and Computer Engineering, Northeastern University
We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.
A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line
1National Center for Natural Products Research, School of Pharmacy, University of Mississippi, 2Department of Pharmacology, University of Mississippi
A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.
Intravascular Perfusion of Carbon Black Ink Allows Reliable Visualization of Cerebral Vessels
Department of Neurology, University of Duisburg-Essen Medical School
Analysis of rodent cerebrovascular anatomy plays an important role in experimental stroke research. In this context, intravascular perfusion with colored latex has been considered as a standard tool for several years. However, this technique implies distinct technical limitations, which undermine its reproducibility. Here, we describe a simple method to visualize cerebral vessels in a reproducible manner. Injection of a mixture of two commercially available carbon black inks through the left myocardial ventricle results in adequate filling of cerebral vessels with high contrast visualization. We have successfully applied this technique to identify anastomotic points between cerebral vascular territories of mice with different genetic backgrounds. We finally give evidence that this novel and simple method for vessel staining can be combined with triphenyltetrazolium chloride (TTC) staining - a widely used tool to observe and analyze infarct volumes in mice.
Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees (Apis mellifera L.)
In this protocol we show how to condition harnessed honey bees to tactile stimuli and introduce a 2D motion capture technique for analyzing the kinematics of fine-scale antennal sampling pattern.
ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells
The biarsenical dyes FlAsH and ReAsH bind specifically to tetracysteine motifs in proteins and can selectively label proteins in live cells. Recently this labeling strategy has been used to develop sensors for different protein conformations or oligomeric states. We describe the labeling approach and methods to quantitatively analyze binding.
Introducing Shear Stress in the Study of Bacterial Adhesion
Blood vessels as a target for infection, Paris center for cardiovascular research, INSERM U970
During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.
Two Types of Assays for Detecting Frog Sperm Chemoattraction
1Department of Animal Sciences, University of Illinois, Urbana-Champaign, 2School of Life Sciences, Arizona State University
Eggs and the extracellular coatings around eggs frequently release peptides, proteins and small molecules that communicate with sperm to guide them to the egg thereby promoting fertilization. Using frog sperm we describe and compare two classes of assays used to detect sperm chemoattraction – sperm accumulation assays and sperm tracking assays.
Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition
1Graduate Group in Biophysics, University of California San Francisco, 2Department of Biochemistry and Biophysics, University of California San Francisco
Visualizing protein samples by negative stain electron microscopy (EM) has become a popular structural analysis method. It is useful for quantitative structural analysis, such as calculating a 3D reconstruction of the molecules being studied, and also for qualitative examination of the quality of protein preparations. In this article we present detailed protocols for preparing the EM grids, staining the sample and visualizing the sample in an electron microscope. Novice users can follow these protocols easily and to utilize negative stain EM as a routine assay, in addition to other biochemical assays, for evaluating their protein samples.
Determining 3D Flow Fields via Multi-camera Light Field Imaging
1Department of Mechanical Engineering, Brigham Young University, 2Naval Undersea Warfare Center, Newport, RI
A technique for performing quantitative three-dimensional (3D) imaging for a range of fluid flows is presented. Using concepts from the area of Light Field Imaging, we reconstruct 3D volumes from arrays of images. Our 3D results span a broad range including velocity fields and multi-phase bubble size distributions.
Near Infrared Optical Projection Tomography for Assessments of β-cell Mass Distribution in Diabetes Research
1Umeå Centre for Molecular Medicine, Umeå University, 2Cell Transplant Center, Diabetes Research Institute, University of Miami,, 3EMBL-CRG Systems Biology Program, Centre for Genomic Regulation, Catalan Institute of Research and Advanced Studies, 4Dept. of Computing Science, Umeå University
We describe the adaptation of optical projection tomography (OPT)1 to imaging in the near infrared spectrum, and the implementation of a number of computational tools. These protocols enable assessments of pancreatic β-cell mass (BCM) in larger specimens, increase the multichannel capacity of the technique and increase the quality of OPT data.
Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth
1Department of Biological Systems Engineering, University of Nebraska-Lincoln, 2Department of Engineering Mechanics, University of Nebraska-Lincoln
The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (μMRE).
Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart
Department of Biomedical Engineering, Washington University in St. Louis
This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.
Tomato Analyzer: A Useful Software Application to Collect Accurate and Detailed Morphological and Colorimetric Data from Two-dimensional Objects
Department of Horticulture and Crop Science, The Ohio State University
Tomato Analyzer (TA) quantifies attributes of two dimensional shapes and color in a reproducible and accurate manner. A step-by-step procedure for obtaining high quality digitalized images of tomato fruit, morphological and color analyses of these images and several applications using the data generated through this software are described.
Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion
Department of Neuroscience and Biomedical Technologies, University of Milano Bicocca
Cerebral perfusion monitoring has been demonstrated to improve accuracy in ischemic stroke models. Technical difficulties often limit the use of this essential tool for cerebrovascular research. In this video, an optimized system is shown to obtain a single or multi-site hemodynamic monitoring during intraluminal middle cerebral artery occlusion in rats.
