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IDDRC: Cellular Imaging and Analysis Core
Overview
The Cellular Imaging and Analysis Core offers advanced optical equipment and computing support that helps investigators observe and analyze biological processes at the tissue, cellular and molecular levels. The core provides advanced microscopes and the technical expertise required to use them for translational research.
The Cellular Imaging and Analysis Core is comprised of two facilities: the Center for Microscopy and Image Analysis (CMIA) located at George Washington University (GWU), and the CRI Light Microscopy and Image Analysis Core (CLIC) located here at Children’s National in the heart of Children’s Research Institute’s Center for Neuroscience Research.
CLIC has an open door policy, providing equipment free-of-charge for all research aimed at understanding and curing intellectual and developmental disabilities. CLIC services and equipment are detailed below.
Services
Services provided by the core include:
- Access to state-of-the-art instruments
- Help with design and execution of experiments
- Hands-on training in optimal use of the equipments by individual users
Equipment
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A composite image of a region of a myofibre of an old dystrophic (Mdx) mouse. Old Mdx mice of around 6 months of age show high numbers of quiescent myoblasts and significant muscle degeneration and regeneration. This myofiber from the Extensor Digitorum Longus muscle of an old dystrophic (Mdx) mouse shows the striated arrangement of costamere contractile proteins in the muscle fiber imaged using the transmitted light, symptomatic 'trains' of centralized nuclei (blue) and quiescent myoblasts at various positions relative to the focal plane (green). Such microscopic analysis of diseased muscle fibers is used to better understand the pathology of Duchene Muscular Dystrophy. The image was acquired using a 40X oil objective on a Nikon Eclipse E500 epifluorescence microscope (Bill Duddy, Center for Genetic Medicine Research).
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Cell Imaging - Equipment available for cell imaging includes:
- Zeiss LSM 510 Confocal Microscope
- Optical sectioning of cells labeled with multiple fluorescent probes allowing multicolor visualization of sub-cellular details in 3D
- Can be equipped with a chamber that permits precise environmental control that allows cells to be cultured and imaged on the microscope for extended periods
- Olympus BX60 Epifluorescence Microscope
- This microscope is equipped with Nomarski/DIC accessories, with an Optronics DEI-750 low-light CCD camera and a FlashPoint 128 video frame grabber
- Allows widefield fluorescence imaging of samples from blue to near infrared fluorescence range
- Zeiss APOTOME Grid Microscope
- Allows routine optical sectioning and live imaging of adherent cells in culture
- Allows users to acquire confocal-like sections with optics for the three most used excitation/emission wavelengths: blue, green, red in addition to the transmitted light detection
- Includes a Zeiss AxioCam MRM CCD camera and high-end computer workstation running Axiovision software
- Olympus Cell Total Internal Reflection Fluorescence (TIRF) System*
- Equipped with Spinning Disc and fluorescence lifetime measurement (FLIM) capability and CCD camera for low-light imaging of live cells
- Capable of imaging live cells and interactions between individual molecules in live cells for custom live cell imaging
* Available through corresponding laboratory
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Cell polarization and death that occurs during the course of lumen formation by human bronchial epithelial cells. Cell nuclei were stained with DAPI (blue). On day 11, the spheroid-like structures were stained to visualize the cell death marker cleaved caspase-8 (green) and the polarization marker integrin α6 (red). The arrows identify cells expressing cleaved caspase. In vitro glands differentiated from respiratory epithelial cells should be useful for the study of respiratory diseases like chronic rhinosinusitis and cystic fibrosis. The image was acquired using optical section of the glandular tissue using LSM510 confocal microscope. (Wu X et al. American Journal of Respiratory Cell and Molecular Biology (2010) 44:1-8.)
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Morphology of two biocytin filled Fast Spiking interneurons in two adjacent barrels of layer 4 in mouse somatosensory cortex. Following injection of Biocytin in neurons in live brain slice, the slice was then fixed and stained with FITC-conjugated Avidin. This image was acquired using LSM510 confocal microscope. Such analysis helps investigate how axons and dendrites of individual neurons project in the brain cortex and the changes that occur in them as a result of developmental brain disorders. (Peijun Li, Center for Neuroscience Research)
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Tissue Imaging - The microscopes capable of high-resolution tissue imaging include:
- Zeiss LSM 510 NLO Microscope
- Equipped with three visible laser lines, three conventional PMT, and a spectral (Meta) detector
- Through the use of Meta detector this laser scanning confocal microscope enables discriminating multiple separate fluorophores with closely overlapping emission spectra
- Coupled to a Verdi 800 pulsed infrared laser that provides multiphoton excitation allowing fluorescence imaging deep into fixed tissues
- Together with equipment for stage incubation and superfusion of organotypic slices this microscope also enables high resolution imaging of live tissues
- Separate offline computer running the full suite of LSM software including deconvolution and 3D modules is networked to the microscope system for file transfers, image analysis, and editing
- C.A.S.T (Computer Assisted Stereological Toolbox) Microscope
- Equipped with motorized stage allowing efficient and unbiased quantitative analysis of number, density, size, and all other stereological quantities of cells in thick tissue specimen
- A Zeiss Stereo LUMAR Microscope
- Available for transmitted, darkfield, morphometric, and fluorescence analyses of mouse embryos and tissue slices
- Equipped with multiple objectives, an AxioCam MRm camera and filters for multicolor fluorescence imaging
- Several Zeiss Stemi 2000 and one Stemi SV11
- Stereo dissecting microscopes are available for gross dissections and the preparation of organotypic slices.
Image Analysis - Core personnel provide individual training and support to help researchers design and carry out image analysis and generate images and quantifications for publications. Several dedicated workstations are available for the offline image analysis needs of researchers. These workstations include the following image analysis software:
- Metamorph premier 7.7
- Volocity
- Image J
- LSM Image Browser
- Matlab
- Loats Image Analysis System: The Loats system is a BM PC/AT video camera based system used for the analysis of biological images that permits the morphological analysis of cells including:
- Measurements of area and perimeter
- Several aspects of shape
- Average and integrated optical densities of immunoreactivity in gels and tissue sections
- Enzyme histochemical densitometric analysis in tissue sections
The System includes:
- IBM PC/AT with a 2.5 MB RAM, a 30 MB hard disk, a 1.2 MB diskette drive, and a serial parallel adapter
- 80287 math coprocessor and an IBM color board
- Rapid digitizer (1/30 second) with a Dage MTI series 68 camera that includes Newvicon tube
- Sony profield monitor for observation of the real time camera image and alphanumeric display
- Mitsubishi monitor for digitized image display
- Leitz Orthoplan microscope equipped with a range of low and high power objectives for dark field and fluorescence microscopy
- Filter box to permit selection of appropriate filters, depending on stains employed
- X Y stage controller including an autofocus (Z axis) with the computer controlling and recording the motion of the stage in all three directions.
Additional Equipment
- Molecular Dynamics PhosphorImager: Utilized for the accurate quantification of labeled macromolecules in gels using Power Macintosh 8100/80av and a 20" color monitor
- Agfa Flatbed and Slide Scanners
- Polaroid Digital Palette Slide Maker
- Tektronix Phaser IIsdx Color Printer
- JEOL JEM-1200EX Transmission Electron Microscope
Equipped with 4 megapixel SIA-CAM camera, via George Washington University Center for Microscopy and Image Analysis
Publications
Aguirre A, Rubio ME, Gallo V. (2010) Notch and EGFR pathway interaction regulates neural stem cell number and self-renewal. Nature, 467: 323-327.
Chakrabarti L, Best TK, Carney RSE, Galdzicki Z, and Haydar TF. (2010) Olig1 and Olig2 triplication causes developmental brain defects in Down syndrome. Nature Neurosci., 13: 927-934.
Etxeberria A, Mangin JM, Aguirre A, Gallo V. Adult-born SVZ progenitors receive transient synapses during remyelination in corpus callosum. Nat Neurosci., 13: 287-289.
Gaddy CE, Wong DS, Markowitz-Shulman A, and Colberg-Poley AM. 2010. Redistribution of the subcellular distribution of key cellular RNA-processing factors during human cytomegalovirus infection. J. Gen. Virology, 91:1547-1559. (Contributed the cover image for this issue)
Hirata T, Li P-J, Lanuza G, Cocas LA, Huntsman MM, and Corbin JG. (2009) Identification of distinct telencephalic progenitor pools for excitatory and inhibitory neuronal cell diversity in the amygdale. Nature Neuroscience, 12:141-149.
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