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.
The following protocol provides techniques for encapsulating pancreatic β-cells in step-growth PEG-peptide hydrogels formed by thiol-ene photo-click reactions. This material platform not only offers a cytocompatible microenvironment for cell encapsulation, but also permits user-controlled rapid recovery of cell structures formed within the hydrogels.
1Interdisciplinary Immunology Program, University of Iowa, and the VA Medical Center, 2Department of Biochemistry, University of Iowa, and the VA Medical Center, 3Department of Internal Medicine, University of Iowa, 4Department of Molecular Microbiology, Washington University School of Medicine, 5Division of Dermatology, Harbor-UCLA Medical Center, Hanley-Hardison Research Center, 6Interdisciplinary Immunology Program, Iowa City VA Medical Center, 7Departments of Internal Medicine, Microbiology and Epidemiology, University of Iowa
An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.
1Department of Biomedical Engineering, University of Wisconsin-Madison, 2Department of Biomedical Engineering, Materials Science Program, Laboratory for Optical and Computational Instrumentation, University of Wisconsin-Madison
A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of ˜1,000 cells in peripheral tissues.
Establishing Intracranial Brain Tumor Xenografts With Subsequent Analysis of Tumor Growth and Response to Therapy using Bioluminescence Imaging
Luciferase-modified human brain tumor xenografts can be established intracranially in athymic mice, with subsequent monitoring of tumor growth and response to therapy using bioluminescence imaging. In combination with survival analysis, bioluminescence monitoring is an essential research tool for pre-clinical testing of therapies being considered for treating brain tumors.
Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.
The method outlines the procedure by which the Hawaiian bobtail squid, Euprymna scolopes and its bacterial symbiont, Vibrio fischeri, are raised separately and then introduced to allow for specific colonization of the squid light organ by the bacteria. Colonization detection by bacterially-derived luminescence and by direct colony counting are described.
This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.
Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.
The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.
Intraductal Injection of LPS as a Mouse Model of Mastitis: Signaling Visualized via an NF-κB Reporter Transgenic
1Cancer Biology Department, Vanderbilt University Medical Center, 2Department of Medicine, Vanderbilt University Medical Center, 3Department of Pharmaceutical Sciences, University of Hawaii at Hilo College of Pharmacy
Described here is a technique in which lipopolysaccharide is injected into the lactating mouse mammary gland via the nipple to simulate mastitis, a condition commonly caused by bacterial infection. Lipopolysaccharide injection results in increased nuclear factor kappa B (NF-κB) signaling, visualized through bioluminescent imaging of an NF-κB luciferase reporter mouse.
Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.
1Department of Medicine, Vanderbilt University School of Medicine, 2Departments of Medicine and Immunology, Roswell Park Cancer Institute, 3Department of Medicine, University at Buffalo School of Medicine
NADPH oxidase is the major source of reactive oxygen species (ROS) in phagocytes. Because of the ephemeral nature of ROS, it is difficult to measure and monitor ROS levels in living animals. A minimally invasive method for serial quantification of ROS in living mice is described.
Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.
To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NFκB.
1Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, 2Brain Research Institute, Molecular Biology Institute, University of California, Los Angeles, 3Department of Neurology, University of California, Los Angeles
Photo-induced cross-linking of unmodified proteins (PICUP) allows characterization of oligomer size distribution in metastable protein mixtures. We demonstrate application of PICUP to three representative amyloidogenic peptides the 40- and 42-residue forms of amyloid β-protein, and calcitonin, and a control peptide growth-hormone releasing factor.
Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy
We describe a method to localize fluorescently tagged proteins in electron micrographs. Fluorescence is first localized using photo-activated localization microscopy on ultrathin sections. These images are then aligned to electron micrographs of the same section.
Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.
Mammary tumor cells expressing luciferase are implanted subcutaneously in mice and visualized using optical imaging to monitor tumor growth and development non-invasively in a longitudinal study.
Micro 3D Printing Using a Digital Projector and its Application in the Study of Soft Materials Mechanics
We demonstrate controlled pattern transformation of swelling gel tubes by elastic instability. A simple projection micro stereo-lithography setup is built using an off-the-shelf digital data projector to fabricate three-dimensional polymeric structures in a layer-by-layer fashion. Swelling hydrogel tubes under mechanical constraint display various circumferential buckling modes depending on dimension.
1Department of Biomedical Engineering, The Ohio State University, 2Department of Biomedical Informatics, The Ohio State University, 3Comprehensive Wound Center, The Ohio State University, 4Department of Surgery, The Ohio State University
A dual-mode imaging system was developed for non-contact assessment of cutaneous tissue oxygenation and vascular function.
This paper illustrates an innovative visual approach (photovoice or photo-elicitation) to achieve fair process in clinical care for patients living with chronic health conditions, illuminate gaps in clinical knowledge, forge better therapeutic relationships, and identify patient-centered goals and possibilities for healing.
This article describes the culture of patient tissue slices for gene delivery studies and subsequent analysis of gene expression using IVIS bioluminescence imaging.
This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.
Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.
A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)
We describe an alternative approach to the enumeration of mycobacteria in vitro, which uses reporter-gene tagged mycobacteria instead of colony-forming units (CFU). “Survival” of organisms as well as host response-markers are measured simultaneously, providing a low-cost, versatile and functional system for studies of host/pathogen interactions in the context of tuberculosis.
Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells
A method for spatio-temporal control of small GTPase activity by light is described. This method is based on rapamycin-induced FKBP-FRB heterodimerization and photo-caging systems. Optimization of light-irradiation enables the spatio-temporally controlled activation of small GTPases at the subcellular level.
A recently developed novel particle agglutination (PA) assay utilizing virus receptor molecule allowed a rapid and easy identification of poliovirus (PV). In this article, we will show the procedure for the PA assay for PV identification.
Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice
1Department of Molecular Microbiology and Immunology, Bond Life Sciences Center, University of Missouri, 2Department of Biological Sciences, Columbia University, 3Department of Veterinary Pathobiology, Bond Life Sciences Center, University of Missouri
This article demonstrates two very different methods of injection: 1) into the brain (intracerebroventricular) and 2) systemic (intravenous) to introduce the therapeutic agents into the central nervous system of neonatal mice.
Analysis of rodent cerebrovascular anatomy plays an important role in experimental stroke research. In this context, intravascular perfusion with colored latex has been considered as a standard tool for several years. However, this technique implies distinct technical limitations, which undermine its reproducibility. Here, we describe a simple method to visualize cerebral vessels in a reproducible manner. Injection of a mixture of two commercially available carbon black inks through the left myocardial ventricle results in adequate filling of cerebral vessels with high contrast visualization. We have successfully applied this technique to identify anastomotic points between cerebral vascular territories of mice with different genetic backgrounds. We finally give evidence that this novel and simple method for vessel staining can be combined with triphenyltetrazolium chloride (TTC) staining - a widely used tool to observe and analyze infarct volumes in mice.
Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.
We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.
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.
1Department of Experimental Oncology, European Institute of Oncology, 2Department of Pathology and Laboratory Medicine, European Institute of Oncology, 3U.O. Gastroenterologia 2, IRCCS Ca' Granda, Ospedale Policlinico di Milano
We introduce a novel method for the maintenance of human intestinal mucosa in culture and monitoring of the response to various types of stimuli over at least 24 hrs. With our method, the polarity of the tissue is maintained, allowing for a physiological stimulation via the apical route.
1Cork Cancer Research Centre, Mercy University Hospital and Leslie C. Quick Jnr. Laboratory, University College Cork, 2Department of Computer Science, University College Cork, 3South Infirmary Victoria University Hospital
This article describes a procedure for the induction of orthotopic bioluminescent liver tumours in mice, and subsequent analysis of tumour growth confined to the liver using live whole body luminescence imaging.
Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging
We demonstrate the metalation, purification, and characterization of lanthanide complexes. The complexes described here can be conjugated to macromolecules to enable tracking of these molecules using magnetic resonance imaging.
Oncolytic viruses are promising for cancer therapeutics. The ability to ascertain the infectability of live tissue specimens obtained from patients prior to treatment is a unique advantage of this therapeutic approach. This protocol describes how to process tissues for ex vivo infection with oncolytic virus and subsequent viral quantification.
Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments
In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium.
Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)
Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.
1Lehrstuhl für Biomolekulare Sensoren, Technische Universität München, 2Center for Integrated Protein Science (Munich) at the Institute of Neuroscience, Technische Universität München, 3TUM Institute for Advanced Study and German Center for Neurodegenerative Diseases, Technische Universität München, 4Munich Cluster for Systems Neurology (SyNergy), Technische Universität München
Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.
American biochemist Roger Tsien shared the 2008 Nobel Prize in Chemistry with Martin Chalfie and Osamu Shimomura for their discovery and development of the Green Fluorescent Protein (GFP). Tsien dramatically improved the wild-type GFP resulting in increased fluorescence, increased photostability, and a shift in the major excitation peak to 488 nm (matching FITC).
A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.
1Department of Physics and Astronomy, The University of Texas at San Antonio, 2Centro de Investigaciones en Optica A. C., 3Department of Biology and Neurosciences Institute, The University of Texas at San Antonio
We synthesized star shaped gold nanostars using a silver seed mediated growth method. The diameter of the nanostars ranges from 200 to 300 nm and the number of tips vary from 7 to 10. The nanoparticles have a broad surface plasmon resonance mode centered in the near infrared.
Patterned Photostimulation with Digital Micromirror Devices to Investigate Dendritic Integration Across Branch Points
Digital micromirror devices (DMD) can generate complex patterns in time and space with which to control neuronal excitability. Issues relevant to the design, construction, and operation of DMD systems are discussed. Such a system enabled the demonstration of non-linear integration across distal dendritic branch points.
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.
Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus
The procedure of preparing slices containing the adult mouse hypothalamic suprachiasmatic nucleus (SCN), and a rapid way to culture the SCN tissue in organotypic culture condition, are reported. Further, the measurement of oscillatory clock gene protein expression using dynamic luciferase reporter technology is described.
Demonstration of key methods for high throughput leaf measurements. These methods can be used to accelerate leaf phenotyping when studying many plant mutants or otherwise screening plants by leaf phenotype.
Aptamers are short ribo-/deoxyribo-oligonucleotides selected by in-vitro evolution methods based on affinity for a specific target. Aptamers are molecular recognition tools with versatile therapeutic, diagnostic, and research applications. We demonstrate methods for selection of aptamers for amyloid β-protein, the causative agent of Alzheimer's disease.
This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.
1Department of Biology, University of Iowa, 2Molecular Targeting Technologies, Inc.
A combination of different techniques to maximize data collection from mouse tissue is presented.