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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
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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
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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 µm3. 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.
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.
- Bushong, E. A., Martone, M. E., et al., Protoplasmic astrocytes in CA1 stratum radiatum occupy
separate anatomical domains. Journal of Neuroscience 22:183–192, 2002.
- Gaietta, G., Deerinck, T., et al., Multicolor and electron microscopic imaging of connectin
traffic. Science 296:503–507, 2002.
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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
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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
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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.
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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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
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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
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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.
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—a package for reconstructing helical macromolecular complexes; Suprim—a general
software package for processing electron microscopy images; Viewit—a multidimensional general
image-processing software package; and XMRBS-Scheduler—a 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.
- 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 137:273282, 2002.
- 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 135:302312, 2001.
- 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 132:3345, 2000.
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New York State Department of Health
Wadsworth Center
Empire State Plaza
Albany, NY 12201-0509
URL: www.wadsworth.org/rvbc
Grant No. P41 RR001219
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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
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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 (“reconstruction engine”). The driving
research projects are the studies of mitochondria, centrosomes, kinetochores, ribosomes, and calcium
release channel.
Instruments
AEI EM7 MKII high-voltage electron microscope (100–1,200 KeV) can image whole-cell mounts or selectively
stained sections >5 µm thick. A JEOL, JEM-4000FX intermediate-voltage electron microscope (IVEM)
(100–4,000 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.
- Wagenknecht, T., Hsieh, C.-E., Rath, B. K., Fleischer, S., and Marko, M., Electron tomography of
frozen-hydrated triad junctions. Biophysical Journal 83:24912501, 2002.
- 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 138:8591, 2002.
- Mannella, C. A., Pfeiffer, D. R., et al., Topology of the mitochondrial inner membrane: Dynamics
and bioenergetic implications. IUBMB Life 52:93100, 2001.
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University of Colorado
Porter Biosciences Building
Boulder, CO 80309-0347
URL:
http://bio3d.colorado.edu
Grant No. P41 RR000592
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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 |
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 (200–500 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.
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 500–750 KeV. This scope uses side entry operation,
grid diameter of 3 mm, magnification range, 150–250,000 X; resolution better than 3 Å
lattice; routine goniometer stage tilting ±60°about any axis; dark field by tilted beam;
electron diffraction camera lengths 1–4 m; 1 K lens-coupled CCD camera. The intermediate-voltage
microscopes cover 80–300 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’s
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.
- 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 98:2399–2406, 2001.
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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
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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
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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.
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.
- 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 99:12155–12160, 2002.
- Ludtke, S. J., Jakana, J., Song, J.-L., Chuang, D., and Chiu, W., A 11.5 Ĺ single particle
reconstruction of GroEL using EMAN. Journal of Molecular Biology 314:253–262, 2002.
- 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 308:1033–1044, 2001.
- 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 8:868–873, 2001.
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