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
JoVE Developmental Biology
1Department of Medical Biophysics, University of Toronto, 2Sunnybrook Research Institute, 3Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto
Here, we present a protocol to inject ultrasound microbubble contrast agents into living, isolated late-gestation stage murine embryos. This method enables the study of perfusion parameters and of vascular molecular markers within the embryo using contrast-enhanced high-frequency ultrasound imaging.
Published March 4, 2015. Keywords: Developmental Biology, Micro-ultrasound, Molecular imaging, Mouse embryo, Microbubble, Ultrasound contrast agent, Perfusion
1Experimental Radiology, Institute of Diagnostic and Interventional Radiology I, Jena University Hospital, 2Department of Pharmaceutical Technology, Friedrich-Schiller-University Jena, 3Center for Electron Microscopy, Jena University Hospital
The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.
Published January 5, 2015. Keywords: Bioengineering, Drug-delivery, Liposomes, Fluorochromes, Fluorescence-quenching, Optical imaging, Inflammation
1Department of Neurology, University of Ulm
Diffusion tensor imaging (DTI) basically serves as an MRI-based tool to identify in vivo the microstructure of the brain and pathological processes due to neurological disorders within the cerebral white matter. DTI-based analyses allow for application to brain diseases both at the group level and in single subject data.
Published July 28, 2013. Keywords: Medicine, Neuroscience, Neurobiology, Molecular Biology, Biomedical Engineering, Anatomy, Physiology, Neurodegenerative Diseases, nuclear magnetic resonance, NMR, MR, MRI, diffusion tensor imaging, fiber tracking, group level comparison, neurodegenerative diseases, brain, imaging, clinical techniques
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 Bioengineering, University of Illinois at Chicago, 2Department of Pathology, University of Illinois at Chicago, 3Department of Biological Sciences, University of Illinois at Chicago, 4Department of Chemistry, University of Illinois at Chicago, 5Department of Nephrology, University of Illinois at Chicago
Fourier Transform Infrared (FT-IR) spectroscopic imaging is a fast and label-free approach to obtain biochemical data sets of cells and tissues. Here, we demonstrate how to obtain high-definition FT-IR images of tissue sections towards improving disease diagnosis.
Published January 21, 2015. Keywords: Medicine, Spectroscopy, Imaging, Fourier Transform, Pathology, Cancer, Liver, Kidney, Hyperspectral, Biopsy, Infrared, Optics, Tissue
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.
Published February 7, 2014. Keywords: Bioengineering, Drosophila melanogaster, Live Imaging, Microfluidics, axonal injury, axonal degeneration, calcium imaging, photoconversion, laser microsurgery
1Department of Diagnostic and Interventional Radiology, University Medical Center, Hamburg, 2Institute of Immunology, University Medical Center, Hamburg, 3University Cancer Center Hamburg, University Medical Center, Hamburg, 4Department of Oncology and Hematology, University Medical Center, Hamburg
This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.
Published April 6, 2015. Keywords: Medicine, Nanobody, antibody, VHH, fluorescence imaging, molecular imaging, xenograft, animal model
1Watson School of Biological Sciences, 2Cold Spring Harbor Laboratory, 3Departments of Medical Genetics, University of Oslo and Oslo University Hospital
We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.
Published March 24, 2013. Keywords: Cancer Biology, Medicine, Molecular Biology, Cellular Biology, Biomedical Engineering, Genetics, Oncology, Pharmacology, Surgery, Tumor Microenvironment, Intravital imaging, chemotherapy, Breast cancer, time-lapse, mouse models, cancer cell death, stromal cell migration, cancer, imaging, transgenic, animal model
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
1Department of Biological Regulation, Weizmann Institute of Science, 2Unit of Biological Services, Weizmann Institute of Science, 3Department of Diagnostic Imaging, Meir Medical Center, 4Pathology Department, Meir Medical Center
We describe how to obtain parametric and vector maps of the diffusion tensor of the breast using magnetic resonance imaging. The protocol and final output following imaging processing are tailored for tracking breast architectural features and detecting breast malignancy.
Published December 15, 2014. Keywords: Medicine, Magnetic Resonance Imaging, breast, breast cancer, diagnosis, water diffusion, diffusion tensor imaging
1Molecular Imaging Laboratory, MGH/MIT/HMS Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital/Harvard Medical School, 2Center for Drug Discovery, School of Pharmacy, China Pharmaceutical University, 3Perkin Elmer
In this video report, we show the application of Cerenkov Luminescence Imaging (CLI) for interscapular brown adipose tissue in mice under activated and depressed conditions.
Published October 7, 2014. Keywords: Medicine, Cerenkov luminescence imaging, brown adipose tissue, 18F-FDG, optical imaging, in vivo imaging, spectral unmixing
1Orthopaedic Hospital Research Center, Orthopaedic Hospital Department of Orthopaedic Surgery, David Geffen School of Medicine at University of California, Los Angeles (UCLA), 2PerkinElmer, 3Department of Dermatology, Johns Hopkins University School of Medicine, 4Department of Medicine, Division of Infectious Diseases, Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine
Combined optical and μCT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.
Published October 16, 2014. Keywords: Infection, imaging, optical, CT, bioluminescence, fluorescence, staphylococcus, infection, inflammation, bone, orthopaedic, implant, biofilm
1Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, 2IBM
The present protocol describes a novel method of identifying a population of enucleating orthochromatic erythroblasts by multi-spectral imaging flow cytometry, providing a visualization of the erythroblast enucleation process.
Published June 6, 2014. Keywords: Basic Protocol, Erythropoiesis, Erythroblast enucleation, Reticulocyte, Multi-Spectral Imaging Flow Cytometry, FACS, Multiparameter high-speed cell imaging in flow, Aspect ratio, Delta centroid XY
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.
Published January 22, 2013. Keywords: Bioengineering, Medicine, Biomedical Engineering, Anatomy, Physiology, Cancer Biology, Pathology, Surgery, Bronchoscopic imaging, In vivo optical microscopy, Optical imaging, Optical coherence tomography, Optical frequency domain imaging, Histology correlation, animal model, histopathology, airway, lung, biopsy, imaging
JoVE Immunology and Infection
1Department of Medicine III, RWTH University-Hospital Aachen, 2IZKF Aachen Core Facility "Two-Photon Imaging", RWTH University-Hospital Aachen, 3Institute for Laboratory Animal Science & Experimental Surgery, RWTH Aachen University, 4Institute for Pharmacology, RWTH University-Hospital Aachen
Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.
Published March 24, 2015. Keywords: Immunology, intravital imaging, TPLSM, two-photon microscopy, liver, migration, microscopy, leukocyte traffic, inflammation
1Department of Neuroscience, Medical University of South Carolina
A method is described for labeling neurons with fluorescent dyes in predetermined functional micro-domains of the neocortex. First, intrinsic signal optical imaging is used to obtain a functional map. Then two-photon microscopy is used to label and image neurons within a micro-domain of the map.
Published December 12, 2012. Keywords: Neuroscience, Molecular Biology, Cellular Biology, Anatomy, Physiology, Two-photon imaging, non-rodent, cortical maps, functional architecture, orientation pinwheel singularity, optical imaging, calcium-sensitive dye, bulk loading, single-cell electroporation
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
JoVE Immunology and Infection
1MRC Centre for Molecular Bacteriology and Infection, Division of Cell & Molecular Biology, Imperial College London, 2Preclinical Imaging, Caliper- A PerkinElmer Company
Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with μCT imaging to create a 4D movie of C. rodentium infection.
Published August 13, 2013. Keywords: Infection, Immunology, Cellular Biology, Molecular Biology, Microbiology, Genetics, Biophysics, Biomedical Engineering, Medicine, Anatomy, Physiology, Infectious Diseases, Bacterial Infections, Bioluminescence, DLIT-μCT, C. rodentium, 4D imaging, in vivo imaging, multi-modality imaging, CT, imaging, tomography, animal model
1Center for Preclinical Imaging, Department of Molecular Biotechnology and Health Sciences, University of Turin, 2Molecular Imaging Center, Department of Molecular Biotechnology and Health Sciences, University of Turin, 3Bracco Research Center, Bracco Imaging SpA
The present work describes a new protocol to perform non-invasive high-frequency ultrasound and photoacoustic based imaging on rat brain, to efficiently visualize deep subcortical regions and their vascular patterns by directing signals on skull foramina naturally present on animal cranium.
Published March 2, 2015. Keywords: Neuroscience, Photoacoustics, High-frequency ultrasounds, Brain imaging, Cerebral hemodynamics, Non-invasive imaging, Small animal, Neuroimaging
1Department of Molecular Genetics and Microbiology, Duke University Medical Center, 2Departments of Neurobiology and Cell Biology, Duke Institute for Brain Sciences, Duke University Medical Center
Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.
Published June 4, 2014. Keywords: Neuroscience, mitosis, radial glial cells, developing cortex, neural progenitors, brain slice, live imaging
1Department of Civil and Environmental Engineering and Earth Sciences, University of Notre Dame, 2Eck Institute for Global Health, University of Notre Dame, 3Department of Applied and Computational Mathematics and Statistics, University of Notre Dame, 4INRS-Institut Armand-Frappier, 5Department of Biology, Indiana University, 6Department of Biological Sciences, University of Notre Dame
Swarming motility is influenced by physical and environmental factors. We describe a two-phase protocol and guidelines to circumvent the challenges commonly associated with swarm assay preparation and data collection. A macroscopic imaging technique is employed to obtain detailed information on swarm behavior that is not provided by current analysis techniques.
Published April 7, 2015. Keywords: Microbiology, Surface motility, Swarming, Imaging, Pseudomonas aeruginosa, Salmonella Typhimurium, Bacillus subtilis, Myxococcus xanthus, Flagella
1University of Victoria-Genome BC Proteomics Centre, University of Victoria, 2Department of Biochemistry and Microbiology, University of Victoria
Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging of small molecules; protocols for the use of DT for the MALDI imaging of endogenous lipids on the surface of tissue sections by positive-ion MALDI-MS on an ultrahigh-resolution quadrupole-FTICR instrument are provided here.
Published November 26, 2013. Keywords: Basic Protocol, eye, molecular imaging, chemistry technique, analytical, mass spectrometry, matrix assisted laser desorption/ionization (MALDI), tandem mass spectrometry, lipid, tissue imaging, bovine lens, dithranol, matrix, FTICR (Fourier Transform Ion Cyclotron Resonance)
1Department of Physiology and Biophysics, Boston University School of Medicine, 2Boston University Photonics Center
With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.
Published April 10, 2013. Keywords: Developmental Biology, Physiology, Biophysics, Neurobiology, Cellular Biology, Molecular Biology, Anatomy, Developmental Biology, Biomedical Engineering, Medicine, Caenorhabditis elegans, C. elegans, Microscopy, Fluorescence, Neurosciences, calcium imaging, genetically encoded calcium indicators, cameleon, GCaMP, neuronal activity, time-lapse imaging, laser ablation, optical neurophysiology, neurophysiology, neurons, animal model
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
1Institute of Bioengineering and Swiss Institute of Experimental Cancer Research (ISREC), École Polytechnique Fédérale de Lausanne, 2Department of Cell and Developmental Biology and Knight Cancer Institute, Oregon Health & Science University
The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.
Published April 22, 2014. Keywords: Bioengineering, Intravital imaging, epifluorescence, two-photon imaging, Tumor matrix, Matrix remodeling
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
1Neurobiology, NCBS-TIFR, 2Department of Biological Sciences, TIFR
A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.
Published September 30, 2012. Keywords: Bioengineering, Molecular Biology, Neuroscience, Microfluidics, C. elegans, Drosophila larvae, zebrafish larvae, anesthetic, pre-synaptic vesicle transport, dendritic transport of glutamate receptors, mitochondrial transport, synaptotagmin transport, heartbeat
1Department of Physics & Astronomy, University of Utah, 2Center for Cell and Genome, University of Utah
The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.
Published January 2, 2014. Keywords: Basic Protocol, AFM, Super-resolution fluorescence imaging, single virion imaging, fPALM, dSTORM, PALM
1Department of Neurosciences, Case Western Reserve University, 2Department of Biomedical Engineering, Case Western Reserve University, 3Department of Pediatrics, Case Western Reserve University
Two-photon intravital imaging can be used to investigate interactions among different cell types in the spinal cord in their native tissue environment in a bone marrow chimeric animal with a dorsal column traumatic spinal cord crush injury.
Published November 23, 2014. Keywords: Cellular Biology, Intravital, spinal cord crush injury, chimera, microglia, macrophages, dorsal column crush, axonal dieback
1Fachbereich Biologie, Entwicklungsbiologie, Philipps-Universität Marburg, 2Institut für Molekularbiologie und Tumorforschung, Philipps-Universität Marburg
This protocol describes the dissection and cultivation of intact testes and germ-line cysts from Drosophila melanogaster pupae. This method allows microscopic observation of spermatogenesis ex vivo. Furthermore, we describe a pharmacological assay of the effect of inhibitors on specific stages of germ-cell development in pupal testes.
Published September 11, 2014. Keywords: Developmental Biology, Ex vivo culture, testis, male germ-line cells, Drosophila, imaging, pharmacological assay
1Department of Surgery, University Medical Center Groningen, 2Helmholtz Zentrum Munich, Technical University of Munich
Near-infrared fluorescence (NIRF) imaging may improve therapeutic outcome of breast cancer surgery by enabling intraoperative tumor localization and evaluation of surgical margin status. Using tissue-simulating breast phantoms containing fluorescent tumor-simulating inclusions, potential clinical applications of NIRF imaging in breast cancer patients can be assessed for standardization and training purposes.
Published September 19, 2014. Keywords: Medicine, Breast cancer, tissue-simulating phantoms, NIRF imaging, tumor-simulating inclusions, fluorescence, intraoperative imaging
JoVE Immunology and Infection
1Center for Molecular Imaging (CMI), University of Texas Health Science Center-Houston
Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.
Published March 8, 2013. Keywords: Immunology, Medicine, Anatomy, Physiology, Molecular Biology, Biomedical Engineering, Cancer Biology, Optical imaging, lymphatic imaging, mouse imaging, non-invasive imaging, near-infrared fluorescence, vasculature, circulatory system, lymphatic system, lymph, dermis, injection, imaging, mouse, animal model
1Neuroscience Center, University of Helsinki
Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.
Published April 6, 2014. Keywords: Medicine, Trauma, Nervous System, animal models, Brain trauma, in vivo multiphoton microscopy, dendrite, astrocyte, microglia, second harmonic generation.
1Institute for Biological and Medical Imaging (IBMI), Helmholtz Zentrum München, 2Faculty of Medicine, Technische Universität München
We provide herein a detailed description of the experimental protocol for imaging with a newly developed hand-held optoacoustic (photoacoustic) system for three-dimensional functional and molecular imaging in real time. The demonstrated powerful performance and versatility may define new application areas of the optoacoustic technology in preclinical research and clinical practice.
Published November 4, 2014. Keywords: Physiology, Optoacoustic tomography, photoacoustic imaging, hand-held probe, volumetric imaging, real-time tomography, five dimensional imaging, clinical imaging, functional imaging, molecular imaging, preclinical research
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
1Institute of Neurobiology, Heinrich Heine University Düsseldorf
We describe the combination of focal UV-induced photo-activation of neuro-active compounds with whole-cell patch-clamp and multi-photon imaging of intracellular sodium transients in dendrites and spines of hippocampal neurons in acute tissue slices of the mouse brain.
Published October 8, 2014. Keywords: Neuroscience, Neurosciences, two-photon microscopy, patch-clamp, UV-flash photolysis, mouse, hippocampus, caged compounds, glutamate, brain slice, dendrite, sodium signals
1Brain Tumor Research Centre, Hospital for Sick Children, Toronto Medical Discovery Tower, 2Ontario Cancer Institute, Princess Margaret Hospital, 3Neurosurgery, Toronto Western Hospital
We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.
Published June 16, 2013. Keywords: Cancer Biology, Medicine, Biomedical Engineering, Cellular Biology, Molecular Biology, Genetics, Neuroscience, Neurobiology, Biophysics, Anatomy, Physiology, Surgery, Intracranial Window, In vivo imaging, Stereotactic radiation, Bone Marrow Derived Cells, confocal microscopy, two-photon microscopy, drug-cell interactions, drug kinetics, brain, imaging, tumors, animal model
1Department of Physics, University of Illinois at Urbana-Champaign, 2Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, 3Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign
Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.
Published September 26, 2014. Keywords: Molecular Biology, FIONA, fluorescence imaging, nanometer precision, myosin walking, thick tissue
1NEST Laboratory, Scuola Normale Superiore, 2Center for Nanotechnology Innovation, Instituto Italiano di Tecnologia, 3Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine
Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.
Published October 9, 2014. Keywords: Bioengineering, fluorescence, protein dynamics, lipid dynamics, membrane heterogeneity, transient confinement, single molecule, GFP
1Neuroscience, Genentech, Inc., 2Department of Discovery Oncology, Genentech, Inc., 3Department of Pathology, Genentech, Inc.
To obtain high-resolution images of fluorescently labeled cells within large tissues, ideally, the biological samples should be imaged without sectioning. 3DISCO is a straightforward tissue clearing procedure based on sequential incubation with organic solvents. Upon clearing, the organs become transparent allowing an end-to-end laser scan of the specimen.
Published July 7, 2014. Keywords: Neuroscience, 3D imaging, tissue clearing, transparent tissue, intact organs, optical clearing, histology, laser scanning, light-sheet microscopy, fluorescent imaging, 3DISCO, ultramicroscope
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 Obstetrics, Gynecology and Reproductive Sciences, Reproductive Immunology Unit, Yale University School of Medicine, 2NatureMost Laboratories, 3Bruker Preclinical Imaging
We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.
Published November 2, 2014. Keywords: Cancer Biology, ovarian cancer, recurrence, in vivo imaging, tumor burden, cancer stem cells, chemotherapy
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
1School of Chemistry and Biochemistry, Georgia Institute of Technology
Desorption electrospray ionization mass spectrometry (DESI-MS) is an ambient method by which samples, including biological tissues, can be imaged with minimal sample preparation. By rastering the sample below the ionization probe, this spray-based technique provides sufficient spatial resolution to discern molecular features of interest within tissue sections.
Published July 12, 2013. Keywords: Bioengineering, Molecular Biology, Biomedical Engineering, Chemistry, Biochemistry, Biophysics, Physics, Cellular Biology, Molecular Imaging, Mass Spectrometry, MS, MSI, Desorption electrospray ionization, DESI, Ambient mass spectrometry, tissue, sectioning, biomarker, imaging
1Department of Neurobiology and Behavior, Cornell University, 2Department of Biomedical Engineering, Cornell University
In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.
Published December 3, 2014. Keywords: Neuroscience, spinal cord, in vivo microscopy, multiphoton microscopy, animal surgery, fluorescence microscopy, biomedical optics
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
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
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 Radiology, Memorial Sloan Kettering Cancer Center
The bioorthogonal inverse electron demand Diels-Alder cycloaddition has been harnessed to create an effective and modular pretargeted PET imaging strategy for cancer. In this protocol, the steps of this methodology are described in the context of a model system employing the colorectal cancer targeted antibody huA33 and a 64Cu-labeled radioligand.
Published February 3, 2015. Keywords: Bioengineering, Positron Emission Tomography, Click Chemistry, Pretargeting, Tetrazine, Trans-cyclooctene, Inverse Electron Demand Diels-Alder Cycloaddition