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Biomedical Technology Resources Directory

Microscopy

 
National Center for Microscopy and Imaging Research
National Center for X-ray Tomography
National Resource for Automated Molecular Microscopy
Resource for the Visualization of Biological Complexity
Three-Dimensional Electron Microscopy of Cells
Three-Dimensional Electron Microscopy of Macromolecules

 
National Center for Microscopy and Imaging Research
University of California, San Diego
1055 Basic Science Building
9500 Gilman Drive
La Jolla, CA 92093-0608

URL: http://gridport.npaci.edu/Telescience
www.nbirn.net

Grant No. P41 RR004050
Principal Investigator and Contact
Mark H. Ellisman, Ph.D.
858-534-2251; Fax: 858-534-7497
E-mail: mark@ncmir.ucsd.edu

Alternate Contacts
Steven Peltier, M.S.
858-534-5637
E-mail: peltier@ncmir.ucsd.edu

Maryann Martone, Ph.D
858-534-8332
E-mail: maryann@ncmir.ucsd.edu

Research Emphasis

The National Center for Microscopy and Imaging Research (NCMIR) specializes in development of technologies to improve the understanding of biological structure and function relationships spanning the dimensional range from 1 nm3 to 50 祄3. Technology development focuses on improving, automating, and providing enhanced access to advanced imaging technologies, including intermediate-high-voltage transmission electron microscopy (IVEM), energy-filtered transmission electron microscopy (TEM) and spectroscopy, large field laser-scanning light microscopy, and real-time (3-D, 4-D) multi-photon microscopy. Through development of the Telescience Portal, the NCMIR features Web-based technologies for collaborative remote access to IVEM, seamless access to distributed computation and data grids, advanced visualization, and integrated access to federated databases to centralize, automate, manage, and accelerate the end-to-end process of electron tomography. The NCMIR is heavily involved in computational biology and information technology; is associated with the Center for Research on Biological Structure at UCSD; and is a satellite site of both the California Institute of Telecommunications and Information Technology and the San Diego Supercomputer Center.

Current Research

Technology development efforts are driven by a range of biological issues in cell and molecular biology, with particular emphasis on neurobiology. Specimen preparation focuses on development of new and improved techniques for selective staining of cells and tissues to facilitate the acquisition of 3-D data using the IVEM. Approaches include 3-D labeling of proteins and nucleic acids within cells and tissues using immunolabeling and non-isotopic in situ hybridization detection, as well as the development of novel nonimmunological protein labeling strategies employing fluorescent compounds combined with molecular biological techniques. Several approaches have been developed for targeting fluorescent probes to specific recombinant proteins followed by photoconversion that renders the same preparation suitable for correlated laser-scanning light microscopy (confocal, multi-photon) and IVEM analysis. The FlAsH-tetracysteine labeling strategy, which relies on the high-affinity covalent bonding of fluorescent bi-arsenical compounds for an engineered short peptide motif, has greatly enhanced the ability to track and localize proteins by both light and electron microscopy. Computer-aided methods are being developed to enhance image contrast and extract accurate 3-D information from within single thick sections or tilt series of sections. Refinement of methods is ongoing for deriving 3-D structure by electron tomography to obtain quantitative structural information on specimens at a variety of scales.

BIRN

The center also is a partner in the Biomedical Informatics Research Network (BIRN) effort of NCRR.

Resource Capabilities

The NCMIR houses three IVEMs specially equipped for electron tomography and two conventional TEMs, one equipped for cryo-electron tomography. A new 300kV centerpiece IVEM features a high-contrast imaging mode, custom optics for high-tilt conical illumination for optical sectioning, an energy filter, a piezo-enhanced high- precision goniometer, three high-resolution digital detectors, an updated "Tele-ready" hardware/software control system, and two laser-scanning confocal microscopes, two real-time multi-photon microscopes, and a physiology station equipped for cell filling.

  1. Bushong, E. A., Martone, M. E., et al., Protoplasmic astrocytes in CA1 stratum radiatum occupy separate anatomical domains. Journal of Neuroscience 22:183�2, 2002.
  2. Gaietta, G., Deerinck, T., et al., Multicolor and electron microscopic imaging of connectin traffic. Science 296:503�7, 2002.
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National Center for X-ray Tomography
University of California, San Francisco
Lawrence Berkeley National Laboratory
Advanced Light Source
1 Cyclotron Rd., MS 6R2100
San Francisco, CA 94720

URL: www.lbl.gov/pbd/ncxt/

Grant No. 1 P41 RR019664-01
Principal Investigator and Contact
Carolyn Larabell, Ph.D.
510-486-5890; Fax: 510-486-5664
E-mail: larabel@itsa.ucsf.edu

Co-investigator:
Mark Le Gros
510-486-6892; Fax: 510-486-5664
E-mail: malegros@lbl.gov

Research Emphasis

The National Center for X-ray Tomography is developing instrumentation and methodology for three-dimensional (3-D) imaging of whole cells up to 10 microns thick. The wavelength of X-rays used (2.4 nm) makes it possible to achieve better resolution than can be obtained with light microscopy, up to 20 nm with the present optics. Biological material is examined at atmospheric pressure and can be fully hydrated and imaged in its native state. Since organic material absorbs approximately an order of magnitude more strongly than water at this wavelength, structural details of cells are easily observed without need for contrast enhancement reagents. Collection of tomographic data sets is fully automated and can be accomplished in less than three minutes, revealing 3-D images of structures throughout the entire cell. Immunolabeling techniques are being developed for 3-D localization of molecules in whole cells. Methods are being developed for high-throughput imaging of dynamic events in live cells using light microscopy and high-resolution analyses of those same cells using X-ray tomography.

Resource Capabilities

The National Center for X-ray Tomography (NCXT) is building a state-of-the-art X-ray microscope dedicated to biological X-ray microscopy and until completed is using the soft X-ray microscope operated by the Center for X-ray Optics (CXRO) located at the Advanced Light Source, LBNL. A fully equipped biological laboratory is available and contains a dedicated cell/tissue culture room and all associated equipment. Rapid freezing devices are also available, including a propane-jet freezer and ethane plunge freezer. Several light microscopes are available, including a Zeiss 510 NLO laser scanning confocal and multiphoton imaging system, a Bio-Rad 1024 confocal microscope, and digital-camera based upright light microscopes. PC and Macintosh computers are available for image processing using Imod, Spider, Image Pro Plus, Amira, and other commercially available programs.

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National Resource for Automated Molecular Microscopy
The Scripps Research Institute
MC CB 129
10550 North Torrey Pines Road
La Jolla, CA 92037

URL: http://nramm.scripps.edu

Grant No. P41 RR017573
Principal Investigator
Bridget Carragher, Ph.D.
858-784-9070; Fax: 858-784-9090
E-mail: bcarr@scripps.edu

Coinvestigator
Clinton S. Potter
858-784-9050; Fax: 858-784-9090
E-mail: cpotter@scripps.edu

Research Emphasis

The overall mission of the National Resource for Automated Molecular Microscopy is to develop, test, and apply technology aimed toward completely automating the processes involved in solving macromolecular structures using cryo-electron microscopy (cryoEM). The goal is to establish a resource to serve as a center for high-throughput molecular microscopy and for transferring this technique to the research community. Core technology development projects include:

Specimen handling: Projects in this core are all related to improving the handling and monitoring of specimens by developing innovative new engineering devices. These include developing methods for high-throughput screening of negatively stained ordered arrays and controlling the stability of cryostage specimen holders. Automated acquisition: The fundamental core technology of the resource is the development of a generalized system for automated image acquisition. Automated processing: The focus of this core is the automation of the data processing that follows image acquisition. A particular emphasis is the automated identification and segmentation of individual structures from electron micrographs. Information handling: The storage, organization, distribution, and archiving of information is the focus of this core project.

Resource Capabilities

Instruments

FEI F20 Tecnai (200 KeV, FEG) equipped with a 2Kx2K Tietz CCD camera; FEI CM200 (200 KeV, FEG) equipped with a 2Kx2K Tietz CCD camera; FEI CM120 (120 KeV, LaB6) equipped with a Gatan video rate camera; FEI EM208 (screening). All of the peripheral equipment normally associated with a cryoEM lab is also available including capabilities for making cryo specimens, cryostages, scanners, an optical bench, etc.

Software

The resource distributes a number of large software packages including Leginon, a system designed to automate the collection of images for cryoEM. A number of smaller software packages related to microscope control and automation are also available: Phoelix梐 package for reconstructing helical macromolecular complexes; Suprim梐 general software package for processing electron microscopy images; Viewit梐 multidimensional general image-processing software package; and XMRBS-Scheduler梐 web-based facilities scheduler.

In addition, a variety of software packages from other sources are available for use at the resource. These include the MRC crystallographic package, Spider, EMAN, O, MatLab, etc.

  1. Fellman, D., Pulokas, J., Milligan, R. A., Carragher, B., and Potter, C. S., A relational database for cryoEM: Experience at one year and 50,000 images. Journal of Structural Biology 137:273–282, 2002.
  2. Zhu, Y., Carragher, B., Kriegman, D. J., Milligan, R. A., and Potter, C. S., Automated identification of filaments in cryoelectron microscopy images. Journal of Structural Biology 135:302–312, 2001.
  3. Carragher, B., Kissebert, N., Kriegman, D., Milligan, R. A., Potter, C. S., Pulokas, J., and Reilein, A., Leginon: An automated system for acquisition of images from vitreous ice specimens. Journal of Structural Biology 132:33–45, 2000.
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Resource for the Visualization of Biological Complexity
New York State Department of Health
Wadsworth Center
Empire State Plaza
Albany, NY 12201-0509

URL: www.wadsworth.org/rvbc

Grant No. P41 RR001219
Principal Investigator
Joachim Frank, Ph.D.
Macromolecular Imaging, Image Processing
518-474-7002; Fax: 518-486-2191
E-mail: joachim@wadsworth.org

Contacts
Carmen A. Mannella, Ph.D.
Electron Tomography
518-474-2462; Fax: 518-402-5381

Terence C. Wagenknecht, Ph.D.
Time-Resolved Imaging
518-474-2450; Fax: 518-474-7992

Research Emphasis

The resource develops methods for the 3-D visualization of biological systems over a wide range of scales, from macromolecular assemblies to cells and tissues. Electron microscopy is the main technique; light microscopy is used to correlate the structural results with cellular-level events.

Current Research

Three areas of technology and research development are pursued: Automated high-resolution low-dose cryoelectron tomography of organelles and cell sections, with the capability to identify macromolecular signatures; time-resolved imaging of macromolecular interactions, using single particle reconstruction methodology; and development of meta-level software for image processing that will allow reconstruction strategies to be formulated (搑econstruction engine�). The driving research projects are the studies of mitochondria, centrosomes, kinetochores, ribosomes, and calcium release channel.

Resource Capabilities

Instruments

AEI EM7 MKII high-voltage electron microscope (100�200 KeV) can image whole-cell mounts or selectively stained sections >5 祄 thick. A JEOL, JEM-4000FX intermediate-voltage electron microscope (IVEM) (100�000 KeV) with a LaB6 cathode, STEM attachment, Gatan GIF2002 energy filter with 2k x 2k cooled CCD camera, 5-axis computer-controlled goniometer, automated data collection, and Gatan cryotransfer stage with tilt-rotate holder. A Philips F20 Technai (200 KeV) with field emission gun and Gatan cryotransfer stage. A Balzers HP-10 high-pressure freezer, and RMC MTX cryo-ultramicrotome. A de Senarmont differential interference contrast-based laser microsurgery/optical-trapping video-enhanced light microscopy workstation (based on Q-switched, pulsed Nd:YAG Surelite II and continuous-wave Nd:YAG 116 EF-CW-3 Quantronix lasers). This low-light level system is capable of near-simultaneous GFP imaging. A Perkin Elmer PDS-1010A flatbed microdensitometer, a Dage/MTI model 81 video camera, and a Eurocore HiScan are used for digitization of images. Several SGI workstations connected to an ONYX 4-processor machine and PC cluster.

Software

SPIDER and associated graphics user interface web, used for all image processing related to single particle reconstruction and tomography. Sterecon, a system to draw contours of stereoscopically represented reconstructions. Explorer, O, Insight, and VoxelView for visualization and interpretation of the results.

Special Features

The IVEM is equipped with single-tilt and tilt-rotation stages for conventional and frozen-hydrated specimens. It is capable of computer-controlled data collection, and both spectrum imaging (using EELS and XMA) and energy-filtered elemental imaging.

  1. Wagenknecht, T., Hsieh, C.-E., Rath, B. K., Fleischer, S., and Marko, M., Electron tomography of frozen-hydrated triad junctions. Biophysical Journal 83:2491–2501, 2002.
  2. Frank, J., Wagenknecht, T., McEwen, B. F., Marko, M., Hsieh, C., and Mannella, C. A., Three-dimensional imaging of biological complexity. Journal of Structural Biology 138:85–91, 2002.
  3. Mannella, C. A., Pfeiffer, D. R., et al., Topology of the mitochondrial inner membrane: Dynamics and bioenergetic implications. IUBMB Life 52:93–100, 2001.
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Three-Dimensional Electron Microscopy of Cells
University of Colorado
Porter Biosciences Building
Boulder, CO 80309-0347

URL: http://bio3d.colorado.edu

Grant No. P41 RR000592
Principal Investigator
J. Richard McIntosh, Ph.D.
303-492-8533; Fax: 303-492-7744
E-mail: richard.mcintosh@colorado.edu

Contact
David Mastronarde, Ph.D.
303-492-4350
E-mail: mast@colorado.edu

Research Emphasis

High-voltage electron microscopy (HVEM) offers scientists the possibility of viewing structural details in specimens whose thickness defies imaging by other forms of EM. This permits three-dimensional (3-D) reconstruction of cellular architecture at resolutions ~40X better than can be achieved by light microscopy. Relevant samples include thick sections (200�0 nm) of cells and tissues or isolated organelles, such as chromosomes, chloroplasts, mitotic spindles, and axonemes.

Stereo images can supply some 3-D information, but more detailed data are available from tomograms calculated from multiple tilted views, e.g., 150 images taken over 140° of tilt about each of two orthogonal axes. The resulting reconstructions show resolution of ~6 nm. This facility is also developing technology for the preparation of reliable cellular specimens. High-quality preservation can be achieved through rapid freezing with or without subsequent freeze-substitution fixation. The resource also is working on specific labeling of macromolecular components of cells, both by conventional immunolabeling and by novel means. Methods for tomographic imaging of frozen hydrated samples are also under development.

Resource Capabilities

Instruments

Microscopes available include a 1 MeV HVEM (JEOL, JEM 1000), as well as 300 and 200 KeV instruments (FEI, F30 and F20) with field emission guns that enhance defocus phase contrast in frozen-hydrated specimens. Equipment for specimen preparation includes plunge and high-pressure freezers, as well as instruments for freeze substitution, low-temperature embedding, and microtomy at temperatures ranging from ambient to liquid nitrogen. Image-processing computers include six fast PCs operating under Linux and two older SGI Octanes.

Special Features

The usable voltage range for the HVEM is 500�0 KeV. This scope uses side entry operation, grid diameter of 3 mm, magnification range, 150�0,000 X; resolution better than 3 Å lattice; routine goniometer stage tilting �°about any axis; dark field by tilted beam; electron diffraction camera lengths 1�m; 1 K lens-coupled CCD camera. The intermediate-voltage microscopes cover 80�0 KeV; both have high-precision goniometer stages and 2K CCD cameras. A Gatan Imaging Filter will soon be added to the F30, together with a lens-coupled 4K CCD camera. The lab抯 image-processing software for 3-D reconstruction on Unix or Linux machines is available for free download from the web site, http://bio3d.colorado.edu.

  1. Marsh, B. J., et al., Organellar relationships in the Golgi region of the pancreatic beta cell line, HIT-T15, visualized by high resolution electron tomography. Proceedings of the National Academy of Sciences USA 98:2399�06, 2001.
Three-Dimensional Electron Microscopy of Macromolecules
National Center for Macromolecular Imaging
Verna and Marrs McLean Department of Biochemistry and Molecular Biology
One Baylor Plaza
Houston, TX 77030

URL: http://ncmi.bcm.tmc.edu

Grant No. P41 RR002250
Principal Investigator and Contactr
Wah Chiu, Ph.D.
713-798-6985; Fax: 713-798-1625
E-mail: wah@bcm.tmc.edu

Coinvestigators
Michael F. Schmid, Ph.D.
713-798-5734; Fax: 713-798-1625
E-mail: mschmid@bcm.tmc.edu

Steven J. Ludtke, Ph.D.
713-798-6989; Fax: 713-798-1625
E-mail: sludtke@bcm.tmc.edu

Research Emphasis

Technology and research development efforts are focused on extending the resolution, speed, and flexibility of electron cryomicroscopy for three-dimensional structure determination of biological macromolecular assemblies. The resource tackles structural problems that are too complex or too difficult for X-ray crystallography and NMR spectroscopy. In the center, researchers have demonstrated the feasibility of visualizing secondary structure elements such as alpha helices and beta sheets of protein components in a number of large assemblies. They are developing technology for routine structure determinations at sub-nanometer resolution, approaching a resolution sufficient for tracing a polypeptide backbone. Generally they focus on macromolecular assemblies ranging from 300 kDa to 30 MDa and can produce structures from very small quantities of purified specimens.

Experimentally, researchers are involved in evaluation of new instruments for single particle imaging, development of automation techniques for high-throughput data collection, and improvements to cryo-preparation techniques. Computationally, they are developing algorithms and improving computational efficiency for the three-dimensional reconstruction of single particles toward atomic resolution. This software is embodied in EMAN and SAVR, which offer complete solutions for low symmetry and icosahedral single particles. In addition, they have produced SAIL, a set of specialized modules for producing professional-quality scientific animations. All three suites and a number of other tools are distributed free of charge.

The majority of efforts are focused on collaborative and service projects with a variety of groups around the world. Current biological projects include cytoskeletal filaments and bundles, ion channels, membrane transporters, icosahedral viruses, and large oligomeric proteins. In addition, the resource sponsors workshops and symposia on a regular basis to disseminate its imaging technology to a broader community.

Resource Capabilities

The resource is uniquely equipped with two high-resolution intermediate-voltage electron cryomicroscopes (JEOL2010F and JEOL3000SFF) operated at liquid nitrogen and at liquid helium temperatures, respectively. Both instruments are equipped with field emission guns and CCD cameras. They have a variety of computational resources, including two PC clusters with an aggregate peak computing power of ~1 teraflop, as well as a variety of PCs, Macs, and SGI workstations. They have shared access to a 32-processor SGI supercomputer with four InfiniteReality graphics heads, used primarily for visualization. The onsite database has ~4 terabytes of online storage for data archival. The National Center for Macromolecular Imaging has an OC-12 link to the Texas Gigapop, with an OC-3 connection to Abilene.

  1. Baker, M. L., Serysheva, I. I., Sencer, S., Wu, Y., Ludtke, S. J., Jiang, W., Hamilton, S. L., and Chiu, W., The skeletal muscle Ca2+ release channel has an oxidoreductase-like domain. Proceedings of the National Academy of Sciences USA 99:12155�160, 2002.
  2. Ludtke, S. J., Jakana, J., Song, J.-L., Chuang, D., and Chiu, W., A 11.5 � single particle reconstruction of GroEL using EMAN. Journal of Molecular Biology 314:253�2, 2002.
  3. Jiang, W., Baker, M. L., Ludtke, S. J., and Chiu, W., Bridging the information gap: Computational tools for intermediate resolution structure interpretation. Journal of Molecular Biology 308:1033�44, 2001.
  4. Zhou, Z. H., Baker, M. L., Jiang, W., Dougherty, M., Jakana, J., Dong, G., Lu, G., and Chiu, W., Electron cryomicroscopy and bioinformatics suggest protein fold models for rice dwarf virus. Nature Structural Biology 8:868�3, 2001.
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