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 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
1Department of Mechanical and Aerospace Engineering, The George Washington University, 2Department of Mechanical and Aeronautical Engineering, Clarkson University
Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient's ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.
Published February 3, 2014. Keywords: Bioengineering, oil-flow visualization, vocal fold polyp, three-dimensional flow separation, aerodynamic pressure loadings
1Institute of Immunology & Experimental Oncology, Center for Biomedical Education and Research (ZBAF), Witten/Herdecke University
Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.
Published October 5, 2014. Keywords: Bioengineering, cell migration, 3D collagen matrix, cell tracking
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
JoVE Clinical and Translational Medicine
1Department of Internal Medicine, University of Michigan Comprehensive Cancer Center, 2Department of Pathology, University of Michigan Comprehensive Cancer Center
Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.
Published April 25, 2014. Keywords: Medicine, pathological conditions, signs and symptoms, neoplasms, three dimensional cultures, Matrigel, breast cells, malignant phenotype, signaling
JoVE Clinical and Translational Medicine
1Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario, 2Department of Oncology, Schulich School of Medicine and Dentistry, University of Western Ontario, 3Lawson Health Research Institute
This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.
Published June 11, 2014. Keywords: Medicine, Breast cancer, cell invasion, extracellular matrix (ECM), three-dimensional (3D) cultures, immunocytochemistry, Matrigel, basement membrane matrix
1Department of Neuroscience, Erasmus MC, 2TNO Human Factors
A method is described to measure three-dimensional vestibulo ocular reflexes (3D VOR) in humans using a six degrees of freedom (6DF) motion simulator. The gain and misalignment of the 3D angular VOR provide a direct measure of the quality of vestibular function. Representative data on healthy subjects are provided
Published May 23, 2013. Keywords: Neurobiology, Neuroscience, Medicine, Anatomy, Physiology, Biomedical Engineering, Ophthalmology, vestibulo ocular reflex, eye movements, torsion, balance disorders, rotation translation, equilibrium, eye rotation, motion, body rotation, vestibular organ, clinical techniques
1Division of Reproductive Sciences, Division of Developmental Biology, Perinatal Institute, Cincinnati Children's Hospital Medical Center, 2Department of Pediatrics, University of Cincinnati College of Medicine
The material here describes a method developed to preserve the three-dimensional chromatin structure of testicular germ cells. This has been termed the three-dimensional (3D) slide method. This method improves sensitivity for detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH).
Published January 10, 2014. Keywords: Basic Protocol, Chromatin, Germ cells, Sex chromosomes, Testis, Meiotic sex chromosome inactivation, Postmeiotic sex chromatin
JoVE Clinical and Translational Medicine
1Department of Neurology, University of Michigan, 2Department of Internal Medicine, University of Michigan
In order to study the changes of nociceptive intraepidermal nerve fibers (IENFs) in painful neuropathies (PN), we developed protocols that could directly examine three-dimensional morphological changes observed in nociceptive IENFs. Three-dimensional analysis of IENFs has the potential to evaluate the morphological changes of IENF in PN.
Published April 29, 2013. Keywords: Medicine, Neurobiology, Neuroscience, Anatomy, Physiology, Cellular Biology, Neurology, Pathology, Peripheral Nervous System Diseases, PNS, Polyneuropathies, Nervous System Diseases, intraepidermal nerve fibers, human skin biopsy, three-dimensional imaging, painful neuropathy, intraepidermal nerve fiber densities, IENFD, nerves, immunohistochemistry, confocal microscopy, imaging
1Department of Materials Science and Engineering, University of Illinois at Urbana-Champaign, 2Center for Micro- and Nanotechnology, Lawrence Livermore National Laboratory, 3Presently at the Interdisciplinary Center for Wide Band-gap Semiconductors, University Of California Santa Barbara
Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.
Published December 9, 2011. Keywords: Bioengineering, Direct-write assembly, silver ink, 3D printing, planar, three-dimensional, microelectrodes, flexible electronics, printed electronics
1University of California, Davis
Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.
Published December 22, 2011. Keywords: Bioengineering, cell invasion, three-dimensional matrix, collagen gel, live-cell confocal imaging, FRAP, GFP, epithelial cyst
JoVE Clinical and Translational Medicine
1Department of Biological Sciences, Purdue University, 2Department of Oncology, University of Alberta, 3Cross Cancer Institute
In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.
Published March 8, 2014. Keywords: Medicine, extracellular matrix, 3D culture, bone marrow, hematological malignancies, primary cell culture, tumor microenvironment
1Molecular Oncology Research Institute, Department of Medicine, Tufts Medical Center
A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.
Published April 30, 2012. Keywords: Molecular Biology, Tumor microenvironment, extracellular matrix, three-dimensional, co-culture, spheroid, mixed-cell, cell culture
JoVE Clinical and Translational Medicine
1Department of Preventive Medicine, University of Southern California, 2Institute for Women's Health, University College London
We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.
Published August 28, 2012. Keywords: Cancer Biology, Medicine, Tissue Engineering, three-dimensional cultures, stromal-epithelial interactions, epithelial ovarian cancer, ovarian surface epithelium, ovarian fibroblasts, tumor initiation
1Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago
Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.
Published June 20, 2011. Keywords: Bioengineering, alginate hydrogel, ovarian organ culture, oviductal organ culture, three-dimensional, primary cell
1Department of Biomedical Engineering, Rensselaer Polytechnic Institute, 2Stemorgan Inc., 3Institute of Advanced Study, Technical University of Munich, 4Institute of Virology, School of Medicine, Wuhan University, 5Department of Molecular and Experimental Medicine, The Scripps Research Institute, 6Research Institute for Biomedical Sciences, Tokyo University of Science
The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.
Published June 10, 2014. Keywords: Bioengineering, cartilage, inkjet printing, chondrocytes, hydrogel, photopolymerization, tissue engineering
JoVE Applied Physics
1Department of Physics, University of California, Irvine, 2Department of Chemistry, University of California, Irvine
The Vaporization of a Sacrificial Component (VaSC) process is used to fabricate microvascular structures. This procedure uses sacrificial poly(lactic) acid fibers to form hollow microchannels with precise 3D geometric positioning provided by laser micromachined guide plates.
Published November 2, 2013. Keywords: Physics, Biomedical Engineering, Chemical Engineering, Silicone Elastomers, Micro-Electrical-Mechanical Systems, Biomimetic Materials, chemical processing (general), materials (general), heat exchangers (aerospace applications), mass transfer, Massive microfabrication, high surface area structures, 3-dimensional micro exchange devices, biomimetics
1Medications Development, Ernest Gallo Clinic and Research Center, University of California, San Francisco, 2Clinical Pharmacology and Experimental Therapeutics, University of California, San Francisco, 3Translational Research Institute and the Institute for Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Australia
We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.
Published August 31, 2012. Keywords: Cellular Biology, 3-dimensional, microscopy, quantification, morphometric, single-cell, cell dynamics
JoVE Applied Physics
13D Machine Vision Laboratory, Department of Mechanical Engineering, Iowa State University
This video describes the fundamentals of digital fringe projection techniques, which provide dense 3D measurements of dynamically changing surfaces. It also demonstrates the design and operation of a high-speed binary defocusing system based on these techniques.
Published December 3, 2013. Keywords: Physics, Structured light, Fringe projection, 3D imaging, 3D scanning, 3D video, binary defocusing, phase-shifting
1Torrey Pines Institute for Molecular Studies, 2Cascade LifeSciences Inc.
The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.
Published July 15, 2013. Keywords: Bioengineering, Cellular Biology, Biophysics, Physiology, Molecular Biology, Biomedical Engineering, Immunology, Cells, Biological Factors, Equipment and Supplies, Cell Physiological Phenomena, Natural Science Disciplines, Life Sciences (General), circulating cells, extravasation, physiological shear stress, endothelial cells, microenvironment, chemokine gradient, flow, chamber, cell culture, assay
1Institute for Biological Interfaces, Forschungszentrum Karlsruhe
We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.
Published May 21, 2008. Keywords: Cellular biology, three-dimensional, chip, bioreactor, perfusion
JoVE Clinical and Translational Medicine
1Raymond and Beverly Sackler Foundation, 2The Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey, 3School of Natural Sciences, Institute for Advanced Study, Princeton, New Jersey
We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.
Published August 14, 2012. Keywords: Cancer Biology, Medicine, Neuroscience, Cell Culture, Tissue Engineering, 3D model, multicellular spheroids, therapeutic drugs, neuroendocrine tumor cell lines, agarose overlay platform, paraffin embedding
1Molecular and Cellular Oncogenesis Program, The Wistar Institute
In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.
Published August 3, 2011. Keywords: Bioengineering, 3D model, melanocyte, melanoma, skin
1Program in Gene Function and Expression, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 2Broad Institute of Harvard and Massachusetts Institute of Technology, 3Division of Health Sciences and Technology, Massachusetts Institute of Technology, 4Program for Evolutionary Dynamics, Department of Organismic and Evolutionary Biology, Department of Mathematics, Harvard University, 5Department of Applied Mathematics, Harvard University, 6Department of Physics, Massachusetts Institute of Technology, 7Department of Systems Biology, Harvard Medical School, 8Department of Biology, Massachusetts Institute of Technology
The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.
Published May 6, 2010. Keywords: Cellular Biology, Chromosome conformation capture, chromatin structure, Illumina Paired End sequencing, polymer physics.
1U.S. Department of Agriculture, 2Department of Viticulture and Enology, University of California - Davis, 3Hawkesbury Institute for the Environment, University of Western Sydney, 4Advanced Light Source, Lawrence Berkeley National Lab, 5Citrus Research & Education Center, University of Florida
High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.
Published April 5, 2013. Keywords: Plant Biology, Cellular Biology, Molecular Biology, Biophysics, Structural Biology, Physics, Environmental Sciences, Agriculture, botany, environmental effects (biological, animal and plant), plants, radiation effects (biological, animal and plant), CT scans, advanced visualization techniques, xylem networks, plant vascular function, synchrotron, x-ray micro-tomography, ALS 8.3.2, xylem, phloem, tomography, imaging
1Department of Radiation Oncology, Virginia Commonwealth University, 2Department of Biochemistry & Molecular Biology, Virginia Commonwealth University, 3Department of Anatomy & Neurobiology, Virginia Commonwealth University, 4Massey Cancer Center, Virginia Commonwealth University
This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.
Published September 28, 2012. Keywords: Genetics, Molecular Biology, Cellular Biology, Biochemistry, DNA, Double-strand breaks, DNA damage response, proteins, live cell imaging, 3D cell imaging, confocal microscopy
1Optical Imaging Laboratory, Department of Biomedical Engineering, Washington University in St. Louis
Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.
Published May 3, 2011. Keywords: Bioengineering, Optical-resolution photoacoustic microscopy, in vivo functional imaging, label-free imaging, noninvasive imaging, hemoglobin oxygen saturation, total hemoglobin concentration
1School of Biomedical Engineering, Science and Health Systems, Drexel University
Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.
Published July 25, 2012. Keywords: Biomedical Engineering, Bioengineering, Biophysics, Cancer Biology, Cancer, interstitial fluid flow, invasion, mechanobiology, migration, three-dimensional cell culture, tumor microenvironment
1Department of Neuroscience & Pharmacology, Rudolf Magnus Institute for Neuroscience, University Medical Center Utrecht
Explants from the midbrain dopamine system and striatum are used in a collagen matrix assay for the in vitro analysis of mesostriatal and striatonigral pathway development. In this assay axonal outgrowth and guidance can be manipulated and quantified. It can also be modified for assessing other regions or molecular cues.
Published March 23, 2012. Keywords: Neuroscience, Axon guidance, collagen matrix, development, dissection, dopamine, medium spiny neuron, rat tail collagen, striatum, striatonigral, mesostriatal
1Institute for Biological Interfaces, Karlsruhe Research Centre, 2Institute for BioMedical Technology, University of Twente, 3Department of Materials Research, Institute for Heavy Ion Research, 4Institute of Microstructure Technology, Karlsruhe Research Centre, 5Institute for Micro Process Engineering, Karlsruhe Research Centre
We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.
Published May 12, 2008. Keywords: Cellular Biology, SMART, microthermoforming, microfabrication, scaffolds, polymer
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
1The Rowland Institute, Harvard University, 2Faculdade de Ciências e Letras de Assis, Universidade Estadual Paulista
The three-dimensional locations of weakly-scattering objects can be uniquely identified using digital inline holographic microscopy (DIHM), which involves a minor modification to a standard microscope. Our software uses a simple imaging heuristic coupled with Rayleigh-Sommerfeld back-propagation to yield the three-dimensional position and geometry of a microscopic phase object.
Published February 8, 2014. Keywords: Basic Protocol, holography, digital inline holographic microscopy (DIHM), Microbiology, microscopy, 3D imaging, Streptococcus bacteria
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.
Published March 22, 2013. Keywords: Medicine, Anatomy, Physiology, Molecular Biology, Biomedical Engineering, Bioengineering, Chemistry, Biochemistry, Materials Science, Engineering, Manufactured Materials, Technology, Animal Structures, Life Sciences (General), 3D printing, X-ray Computed Tomography, CT, CT scans, data extrusion, additive printing, in vivo imaging, clinical techniques, imaging
JoVE Application Notes
In vitro mammalian cell culture has served as an invaluable tool in cell biology for several decades. Classically, monolayer cultures of adherent cells were grown on flat and rigid two-dimensional (2D) substrates, such as polystyrene or glass. However, many cells, when isolated from tissues and placed onto stiff planar 2D cell culture surfaces, such as tissue culture plastic, become progressively flatter, divide aberrantly, and lose their differentiated phenotype1,2. While these two-dimensional cell culture studies have played a pivotal role in furthering our understanding of many biological processes, they do not emulate in vivo conditions.
Published October 16, 2014. Keywords:
1New Jersey Neuroscience Institute, JFK Medical Center, 2Department of Biomedical Engineering, Rutgers University, 3Department of Mechanical and Aerospace Engineering, Rutgers University
Our laboratory has developed DNA-crosslinked polyacrylamide hydrogels, a dynamic hydrogel system, to better understand the effects of modulating tissue stiffness on cell function. Here, we provide schematics, descriptions, and protocols to prepare these hydrogels.
Published August 27, 2014. Keywords: Bioengineering, bioengineering (general), Elastic, viscoelastic, bis-acrylamide, substrate, stiffness, dynamic, static, neuron, fibroblast, compliance, ECM, mechanobiology, tunable
JoVE Clinical and Translational Medicine
1Division of Computer-assisted Restorative Dentistry, Center of Dental Medicine, University of Zürich
Accuracy is a major demand in dental medicine. To verify accuracy, reference scanners are needed. This article presents a new reference scanner with an adjusted scanning method to acquire a broad variety of dental morphologies with high trueness and precision.
Published April 29, 2014. Keywords: Medicine, Laboratories, Dental, Calibration, Technology, Dental impression, Accuracy, Trueness, Precision, Full arch scan, Abrasion
1Basic Medical Sciences, University of Arizona College of Medicine - Phoenix
A rotating cell culture system that allows epithelial cells to grow under physiological conditions resulting in 3-D cellular aggregate formation is described. The aggregates generated display in vivo-like characteristics not observed in conventional culture models and serve as a more accurate organotypic model system for a multitude of scientific investigations.
Published April 3, 2012. Keywords: Cellular Biology, Rotating wall vessel bioreactor, female reproductive tract, human epithelial cells, three-dimensional in vitro cell culture, organotypic mucosal models, vaginal epithelial cells, microbicide, herpes simplex virus, toxicology, host-pathogen interactions, hormone receptors
1Biomedical Engineering Department, Georgia Institute of Technology
An adhesion frequency assay for measuring receptor-ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells is described. This mechanically-based assay is exemplified using a micropipette-pressurized human red blood cell as adhesion sensor and integrin αLβ2 and intercellular adhesion molecule-1 as interacting receptors and ligands.
Published November 2, 2011. Keywords: Bioengineering, Two-dimensional binding, affinity and kinetics, micropipette manipulation, receptor-ligand interaction
1Department of Chemistry and Biochemistry, University of Notre Dame, 2Harper Cancer Research Institute, University of Notre Dame
Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.
Published December 5, 2014. Keywords: Bioengineering, 3D cell culture, mass spectrometry, imaging, cell culture, sample preparation, spheroids
1Life Sciences Division, Lawrence Berkeley National Laboratory, 2Joint Bioenergy Institute, Physical Biosciences Division, Lawrence Berkeley National Laboratory, 3National Energy Research Scientific Computing Center, Lawrence Berkeley National Laboratory
The bottleneck for cellular 3D electron microscopy is feature extraction (segmentation) in highly complex 3D density maps. We have developed a set of criteria, which provides guidance regarding which segmentation approach (manual, semi-automated, or automated) is best suited for different data types, thus providing a starting point for effective segmentation.
Published August 13, 2014. Keywords: Bioengineering, 3D electron microscopy, feature extraction, segmentation, image analysis, reconstruction, manual tracing, thresholding
1Molecular 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).
Published March 28, 2011. Keywords: Structural Biology, Electron microscopy, single particle three-dimensional reconstruction, exosome complex, negative staining
JoVE Applied Physics
1Institute of Applied Research, Aalen University
A module for single plane illumination microscopy (SPIM) is described which is easily adapted to an inverted wide-field microscope and optimized for 3-dimensional cell cultures. The sample is located within a rectangular capillary, and via a microfluidic system fluorescent dyes, pharmaceutical agents or drugs can be applied in small quantities.
Published August 15, 2014. Keywords: Physics, Fluorescence, light sheet, single plane illumination microscopy (SPIM), 3D cell cultures, rectangular capillary, microfluidics, multi-cellular tumor spheroids (MCTS), wide-field microscopy
1Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, 2Department of Chemistry, The Johns Hopkins University
We describe experimental details of the synthesis of patterned and reconfigurable particles from two dimensional (2D) precursors. This methodology can be used to create particles in a variety of shapes including polyhedra and grasping devices at length scales ranging from the micro to centimeter scale.
Published February 4, 2013. Keywords: Chemistry, Chemical Engineering, Biomolecular Engineering, Materials Science, Physics, Nanotechnology, Molecular Self-assembly, Electrochemistry, Folding, three dimensional, lithography, colloid, patchy particles, particles, nanoparticles, robotics, drug delivery, microfabrication, nanofabrication, nano, assembly, synthesis, reaction, origami
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.
Published May 20, 2014. Keywords: Chemistry, confocal microscopy, particle tracking, colloids, suspensions, confinement, gelation, microfluidics, image correlation, dynamics, suspension flow
JoVE Applied Physics
1Department of Physics and Astronomy, University College London, 2CERN, 3Physics Division, Lawrence Berkeley National Laboratories
This paper demonstrates a protocol for recasting experimental simplified model limits into conservative and aggressive limits on an arbitrary new physics model. Publicly available LHC experimental results can be recast in this manner into limits on almost any new physics model with a supersymmetry-like signature.
Published November 15, 2013. Keywords: Physics, high energy physics, particle physics, Supersymmetry, LHC, ATLAS, CMS, New Physics Limits, Simplified Models
1DNA Medicine Institute, 2Harvard Medical School, 3NASA Glenn Research Center, 4ZIN Technologies
Spaceflight blood diagnostics need innovation. Few demonstrations have been published illustrating in-flight, reduced-gravity health diagnostic technology. Here we present a method for construction and operation of a parabolic flight test rig for a prototype point-of-care flow-cytometry design, with components and preparation strategies adaptable to other setups.
Published November 13, 2014. Keywords: Cellular Biology, Point-of-care, prototype, diagnostics, spaceflight, reduced gravity, parabolic flight, flow cytometry, fluorescence, cell counting, micromixing, spiral-vortex, blood mixing
1Analytical Science Division, National Physical Laboratory
We describe the preparation of three test samples and how they can be used to optimize and assess the performance of STORM microscopes. Using these examples we show how to acquire raw data and then process it to acquire super-resolution images in cells of approximately 30-50 nm resolution.
Published September 6, 2013. Keywords: Molecular Biology, Genetics, Bioengineering, Biomedical Engineering, Biophysics, Basic Protocols, HeLa Cells, Actin Cytoskeleton, Coated Vesicles, Receptor, Epidermal Growth Factor, Actins, Fluorescence, Endocytosis, Microscopy, STORM, super-resolution microscopy, nanoscopy, cell biology, fluorescence microscopy, test samples, resolution, actin filaments, fiducial markers, epidermal growth factor, cell, imaging
1Laboratoire Interfaces et Fluides Complexes, Université de Mons
We present a new polyacrylamide hydrogel, called hydroxy-PAAm, that allows a direct binding of ECM proteins with minimal cost or expertise. The combination of hydroxy-PAAm hydrogels with microcontact printing facilitates independent control of many cues of the natural cell microenvironment for studying cellular mechanostransduction.
Published August 28, 2014. Keywords: Bioengineering, hydrogels, mechanotransduction, polyacrylamide, microcontact printing, cell shape, stiffness, durotaxis, cell-ligand density
JoVE Immunology and Infection
1Department of Health Science & Technology, Cartilage Engineering & Regeneration, 2Biomaterials Department, Innovent e.V.
A bioprinter was used to create patterned hydrogels based on a sacrificial mold. The poloxamer mold was backfilled with a second hydrogel and then eluted, leaving voids which were filled with a third hydrogel. This method uses fast elution and good printability of poloxamer to generate complex architectures from biopolymers.
Published July 10, 2013. Keywords: Bioengineering, Immunology, Cellular Biology, Biomedical Engineering, Biophysics, Molecular Biology, Materials Science, Tissue Engineering, Biomaterials, Hydrogel, Biopolymers, Structured/Patterned Hydrogels, Bioprinter, Sacrificial Mold, Thermoresponsive Polymers, Poloxamer, tissue, polymer, matrix, cell, cell culture