1NMR Surgical Laboratory, Department of Surgery, Massachusetts General Hospital, Harvard Medical School, 2Shriners Burn Institute, 3Department of Radiology, Athinoula A. Martinos Center of Biomedical Imaging, Harvard Medical School, 4Molecular Surgery Laboratory, Department of Surgery, Massachusetts General Hospital, Harvard Medical School
This technique enables the use of high-resolution magic angle spinning proton MR spectroscopy (HRMAS 1H-MRS) for molecular characterization of live Drosophila melanogaster with a conventional 14.1 tesla spectrometer equipped with an HRMAS probe.
Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy
A progressive colonization procedure is described to further assess its impact on the host hepatic metabolism. Colonization is monitored non invasively by evaluating the urinary excretion of microbial co-metabolites by NMR-based metabolic profiling while hepatic metabolism is assessed by High Resolution Magic Angle Spinning (HR MAS) NMR profiling of intact biopsy.
1Institute for Solid State Research, IFW-Dresden, 2Institute of Metal Physics of National Academy of Sciences of Ukraine, 3Diamond Light Source LTD, 4Department of Physics, University of Johannesburg, 5CNR-SPIN, and Dipartimento di Fisica "E. R. Caianiello", Università di Salerno, 6Institute of Physics of Complex Matter, École Polytechnique Fédérale de Lausanne
The overall goal of this method is to determine the low-energy electronic structure of solids at ultra-low temperatures using Angle-Resolved Photoemission Spectroscopy with synchrotron radiation.
Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy
1Biosphere Oriented Biology Research Unit, RIKEN Advanced Science Institute, 2Graduate School of Nanobioscience, Yokohama City University, 3Advanced NMR Metabomics Research Team, RIKEN Plant Science Center, 4Graduate School of Bioagricultural Science, Nagoya University
A method for metabolite extraction from microbial planktonic communities is presented. Whole community sampling is achieved by filtration onto specially prepared filters. After lyophilization, aqueous-soluble metabolites are extracted. This approach allows for application of environmental metabolomics to trans-omics investigations of natural or experimental microbial communities.
1Department of Chemistry, City College of New York, City University of New York Graduate Center and Institute for Macromolecular Assemblies, 2Department of Chemical Engineering, City College of New York
Aerial plant organs are protected by the cuticle, a supramolecular biopolyester-wax assembly. We present protocols to monitor selective removal of epi- and intracuticular waxes from tomato fruit cuticles on molecular and micro scales by solid-state NMR and atomic force microscopy, respectively, and to assess the cross-linking capacity of engineered cuticular biopolyesters.
Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices
An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.
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.
Dry Oxidation and Vacuum Annealing Treatments for Tuning the Wetting Properties of Carbon Nanotube Arrays
This article describes a simple method to fabricate vertically aligned carbon nanotube arrays by CVD and to subsequently tune their wetting properties by exposing them to vacuum annealing or dry oxidation treatment.
Despite the outstanding mechanical and biochemical properties of spider silks, this material cannot be harvested in large quantities by conventional means. Here we describe an efficient strategy to spin artificial spider silk fibers, which is an important process for investigators studying spider silk production and their use as next-generation biomaterials.
Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and and Induced Pluripotent Stem Cells (iPSCs)
This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.
Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM)
Topology of cell adhesion on a substrate is measured with nanometre precision by variable-angle total internal reflection fluorescence microscopy (VA-TIRFM).
Femtosecond-laser direct-writing is frequently used to create three-dimensional (3D) patterns in polymers and glasses. However, patterning metals in 3D remains a challenge. We describe a method for fabricating silver nanostructures embedded inside a polymer matrix using a femtosecond laser centered at 800 nm.
Biomaterials Research Group, University of Bayreuth
Spider silk fibers display extraordinary mechanical properties. Engineered Araneus diadematus Fibroin 4 (eADF4) can be processed into nonwoven meshes using electrospinning. Here, the eADF4 nonwoven meshes are used to improve the performance of air filtering devices.
A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.
1Department of Biomedical Science, Cornell University, 2Department of Ecology and Evolutionary Biology, Cornell University, 3Cornell University Museum of Vertebrates, 4Department of Computer Science, Cornell University
We present a non-destructive method for sampling spatial variation in the direction of light scattered from structurally complex materials. By keeping the material intact, we preserve gross-scale scattering behavior, while concurrently capturing fine-scale directional contributions with high-resolution imaging. Results are visualized in software at biologically-relevant positions and scales.
Slice shear force is a reference method for beef texture analysis. Using an angle adjustable cutting box could increase its accuracy for research purposes. The results from different locations within the longissimus muscle show a high correlation with Warner-Bratzler shear force methodology and high potential adaptability for different muscles.
The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures.
A methodology to estimate ventricular fiber orientations from in vivo images of patient heart geometries for personalized modeling is described. Validation of the methodology performed using normal and failing canine hearts demonstrate that that there are no significant differences between estimated and acquired fiber orientations at a clinically observable level.
Use of photonic crystal slow light waveguides and cavities has been widely adopted by the photonics community in many differing applications. Therefore fabrication and characterization of these devices are of great interest. This paper outlines our fabrication technique and two optical characterization methods, namely: interferometric (waveguides) and resonant scattering (cavities).
This protocol describes a method for micron-scale three-dimensional imaging of oxygen concentration in the immediate environment of live cells by electron spin resonance microscopy.
Retraining abnormal movement patterns following injury or disease is a key component of physical rehabilitation. Recent advances in technology have permitted accurate assessment of movement during a variety of tasks, with near instantaneous quantification of results. This provides new opportunities for modification of faulty movement patterns in real time.
We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.
The production of hyperpolarized xenon by means of spin exchange optical pumping (SEOP) is described. This method yields a ~10000-fold enhancement of the nuclear spin polarization of Xe-129 and has applications in nuclear magnetic resonance spectroscopy and imaging. Examples of gas phase and solution state experiments are given.
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.
This protocol outlines the simulation, fabrication and characterization of THz metamaterial absorbers. Such absorbers, when coupled with an appropriate sensor, have applications in THz imaging and spectroscopy.
The following protocol outlines the process of pancreatic dissection for virtual slice imaging, and the subsequent quantification of all GFP-tagged beta-cells in the entire pancreas.
A method to measure the persistence length or flexural rigidity of biopolymers is described. The method uses a kinesin-driven microtubule gliding assay to experimentally determine the persistence length of individual microtubules and is adaptable to actin-based gliding assays.
Here we describe an optimized technique to produce high-quality vitamin A/RBP complex and two real-time monitoring techniques to study vitamin A transport by STRA6, the RBP receptor.
Characterization of Surface Modifications by White Light Interferometry: Applications in Ion Sputtering, Laser Ablation, and Tribology Experiments
White light microscope interferometry is an optical, noncontact and quick method for measuring the topography of surfaces. It is shown how the method can be applied toward mechanical wear analysis, where wear scars on tribological test samples are analyzed; and in materials science to determine ion beam sputtering or laser ablation volumes and depths.
Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps
Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.
Magnetically-Assisted Remote Controlled Microcatheter Tip Deflection under Magnetic Resonance Imaging
1Department of Radiology and Biomedical Imaging, University of California, San Francisco, 2School of Medicine, University of California, San Francisco, 3Department of Radiology and Biomedical Imaging, UCSF Medical Center, 4University of California, San Francisco, 5Hansen Medical, Mountain View, CA
Current applied to an endovascular microcatheter with microcoil tip made by laser lathe lithography can achieve controllable deflections under magnetic resonance (MR) guidance, which may improve speed and efficacy of navigation of vasculature during various endovascular procedures.
1Department of Anatomy and Structural Biology, Albert Einstein College of Medicine - Yeshiva University, 2Gruss Lipper Biophotonics Center, Albert Einstein College of Medicine - Yeshiva University, 3Hubrecht Institute-KNAW and University Medical Center Utrecht
Intravital photoswitching and tracking of Dendra2-labeled tumor cells through the Mammary Imaging Window is a technique which allows us to image the metastatic behavior of tumor cells in chosen tumor microenvironments over a timescale of days.
Described here is an in vivo technique to image sub-cellular structures in animals exposed to anoxia using a gas flow through microincubation chamber in conjunction with a spinning disc confocal microscope. This method is straightforward and flexible enough to suit a variety of experimental parameters and model systems.
1Caltech Optical Observatories, California Institute of Technology, 2Department of Astronomy, California Institute of Technology, 3Dunlap Institute for Astronomy and Astrophysics, University of Toronto, 4Inter-University Centre for Astronomy & Astrophysics, 5Observatories of the Carnegie Institution for Science, 6Benoziyo Center for Astrophysics, Weizmann Institute of Science
Light from astronomical objects must travel through the earth's turbulent atmosphere before it can be imaged by ground-based telescopes. To enable direct imaging at maximum theoretical angular resolution, advanced techniques such as those employed by the Robo-AO adaptive-optics system must be used.
Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time
1Diabetes Research Institute, University of Miami Miller School of Medicine, 2Department of Surgery, University of Miami Miller School of Medicine, 3Department of Medicine, University of Miami Miller School of Medicine, 4Department of Physiology & Biophysics, University of Miami Miller School of Medicine, 5The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet
A new approach combining intraocular transplantation and confocal microscopy enables longitudinal, non-invasive real-time imaging with single-cell resolution within grafted tissues in vivo. We demonstrate how to transplant pancreatic islets into the anterior chamber of the mouse eye.
Demonstration of quantification of dsDNA using Molecular Probes PicoGreen dye and Hitachi F-7000 Fluorescence Spectrophotometer equipped with a microplate reader accessory.
In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.
1Dental and Craniofacial Research Institute and Section of Orthodontics, School of Dentistry, UCLA, 2UCLA and Orthopaedic Hospital, Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, UCLA, 3Department of Bioengineering, UCLA, 4Center for Cardiovascular Science, University of Edinburgh
Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.
The demonstration of the small and wide angle X-ray scattering (SWAXS) procedure has become instrumental in the study of biological macromolecules. Through the use of the instrumentation and procedures of specific angle methods and preparation, the experimental data from the SWAXS displays the atomic and nano-scale characterization of macromolecules.
Bioscience Division, High Content Analysis Research and Development, Millipore Inc
Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma
1Department of Neurobiology and Anatomy, Program in Cancer Cell Biology, Mary Babb Randolph Cancer Center, West Virginia University, 2Sensory Neuroscience Research Center, West Virginia University, 3Departments of Otolaryngology and Physiology, Center for Neuroscience, West Virginia University
A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.
We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.
Nanomoulding of Functional Materials, a Versatile Complementary Pattern Replication Method to Nanoimprinting
1Institute of Microengineering (IMT), Photovoltaics and Thin Film Electronics Laboratory, Ecole Polytechnique Fédérale de Lausanne (EPFL), 2Department of Electrical Engineering and Computer Sciences, University of California, Berkeley
We describe a nanomoulding technique which allows low-cost nanoscale patterning of functional materials, materials stacks and full devices. Nanomoulding can be performed on any nanoimprinting setup and can be applied to a wide range of materials and deposition processes.
Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution
1Electrical Engineering Department, University of California, Los Angeles, 2Bioengineering Department, University of California, Los Angeles, 3California NanoSystems Institute, University of California, Los Angeles
Lensfree optical tomography is a three-dimensional microscopy technique that offers a spatial resolution of <1 μm × <1 μm × <3 μm in x, y and z dimensions, respectively, over a large imaging-volume of 15-100 mm3, which can be particularly useful for integration with lab-on-a-chip platforms.
Quantitative Fitness Analysis (QFA) is a complementary series of experimental and computational methods for estimating microbial culture fitnesses. QFA estimates the effect of genetic mutations, drugs or other applied treatments on microbe growth. Experiments scaling from focussed analysis of single cultures to thousands of parallel cultures can be designed.
This product demonstration video, from Leica Microsystems and myNeurolab.com, illustrates usage of the innovative Leica Angle Two™ computer-guided stereotaxic instrument for mouse, rat and other lab animals. The Angle Two™ allows neuroscientists to be more precise and repeatable in targeting specific brain locations; thus reducing animal costs. Charles Scouten, PhD, Product & Innovation Manager at Leica Microsystems, guides the viewer step-by-step through the usage of this unique stereotaxic instrument.
In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.
Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.
Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate
1Department of Surgery, Stanford University, 2Department of Surgery, Duke University, 3Department of Surgery, Saint Joseph Mercy Hospital, 4School of Medicine, University of California, San Francisco, 5School of Dentistry, University of California, Los Angeles
This protocol describes the isolation of adipose-derived stromal cells from lipoaspirate and the creation of a 4 mm critical-sized calvarial defect to evaluate skeletal regeneration.
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.