National Center for Microscopy and Imaging Research

National Center for Microscopy and Imaging Research

Research Emphasis

A key emphasis of the National Center for Microscopy and Imaging Research (NCMIR) is the application of advanced imaging technologies to the nervous system in health and disease. While maintaining a focus on neurobiology, the NCMIR also addresses the cellular and molecular underpinnings of biological systems relevant to cancer, diabetes, and heart disease. Affiliated with the University of California San Diego's Center for Research in Biological Systems (CRBS), the NCMIR is a recognized authority in the development of technologies for high throughput multiscale imaging and analysis of biological systems at the mesoscale, the dimensional range spanning 5 nm³ and 50 µm³. Macromolecules, organelles, and multicomponent structures such as synapses, which are encompassed in this dimensional range, have traditionally been challenging to study because they fall in the resolution gap between X-ray crystallography at one end and medical imaging at the other.

The NCMIR develops tools to bridge the gaps between technologies, helping hundreds of researchers to make observations at the supramolecular scales that have remained most challenging for biologists. The mission to serve this dimensional range has led to the continued development of state-of-the-art three-dimensional (3-D) imaging and analysis technologies as well as a myriad of innovations in software systems for instrument automation and control, collaboration,databases, and informatics.

The NCMIR's development efforts focus 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 and four dimensional (4-D) multi and photon microscopy. The NCMIR is also continuously working to improve Web-based technologies for collaborative remote access to IVEMs, seamless access to distributed computation and data grids, advanced visualization, and integrated access to federated databases through the Telescience Portal. The Telescience environment allows researchers to build an end-to-end process for automating and accelerating electron tomography workflows. The Telescience technology has also served as the springboard for many leading cyberinfrastructure projects, including the Biomedical Informatics Research Network (BIRN). NCMIR's internationally recognized emphasis on interdisciplinary science brings together local, national, and international researchers from academia, industry, and government; biologists, chemists, physicists, computer scientists, and engineers collaborate at the NCMIR to advance biomedical research.

Current Research

The NCMIR's research focuses on biological specimen development, instrument development, and software infrastructure development. These three core areas maximize the advantages of higher-voltage electron microscopy and correlated light microscopies to make ambitious imaging studies across scales routine and to facilitate the use of resources by biomedical researchers. The NCMIR actively recruits outside users who will not only make use of these resources, but who also will drive technology development and receive training across three core areas:

Biological specimen development for IVEMs and correlated microscopies: The NCMIR is building on continued success in developing contrast-enhancing methods and correlated light and electron microscopies for large-scale imaging, which span the dimensional range and scale of tissue organization to macromolecular specializations of cellular microdomains.

Imaging instrumentation, including IVEM, camera development, stage development, and telemicroscopy: Responding to the increasing emphasis on multiscale integration in biological systems, the NCMIR is expanding its activities in correlated microscopy, integrating multiphoton light and ultra high-voltage electron microscopic techniques to accommodate collaborative multiscale studies. To support ultra wide-field imaging, the NCMIR is building on the success of its ultra large-field lens-coupled charge-coupled device (CCD) camera technologies, pushing the resolution and performance of state-of-the-art digital detectors for high-voltage electron microscopy.

Advanced software infrastructure applications and database development: The NCMIR is making significant contributions in the area of grid-enabled environments for end-to-end electron tomography with the development of its Telescience Portal. Many applications have been modified for Web-based operation within a globally distributed grid environment. The Cell Centered Database, a sophisticated Web-based database, is a data management tool for the resource and a means to disseminate and share data produced at the NCMIR.

Resource Capabilities

Microscope systems: The NCMIR houses three IVEMs specially equipped for electron tomography and three conventional TEMs, one equipped for cryoelectron tomography. A new 300-keV centerpiece IVEM features a high-contrast imaging mode, custom optics to enable 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, two laser-scanning confocal microscopes, one high resolution multiphoton microscope, two real-time multiphoton microscopes, one spinning disk confocal microscope, and a physiology station equipped for cell filling. Detailed descriptions of these resources are described under Instruments.

Computer facilities at the NCMIR feature two computational clusters, >12 tetrabytes (TB) of networked storage, and a network of instrumentation resources, computational resources, and desktop computers linked to other research networks and the Internet. Integrated multiple-processor workstations with up to 32 gigabytes of random access memory support image processing and visualization operations. A 32-node Pentium III cluster and a 21-node Opteron visualization cluster are available to address larger computational challenges. The Telescience Portal links local resources with powerful remote computational and storage resources, including the OptIPuter. National Science Foundation-sponsored OptIPuter is an Internet protocol-based computing platform in which dynamically controllable optical networks enable scientists to interactively visualize, analyze, and correlate their data from distributed sites.

Instruments

IVEMs
JEM-3200EF IVEM (300 kV): Features custom optics for high-tilt conical illumination for optical sectioning; an energy filter; a piezo-enhanced, high-precision goniometer; three high-resolution digital detectors; and an updated hardware/software control system compatible with the Telescience Portal.

JEM-4000EX IVEM (400 kV): The NCMIR has two of these instruments (a production instrument and a test/development resource). Both instruments are specially equipped for electron tomography. The production instrument features a high-contrast imaging mode and custom optics for high-tilt conical illumination for optical sectioning and is equipped for remote control via Telescience.

Ultra High-Voltage Electron Microscope (UHVEM)
Hitachi H-3000 UHVEM at Osaka University (3.0 MeV): The NCMIR houses an exclusive remote kiosk for controlling the world's largest transmission electron microscope. This unique instrument is equipped for electron tomography and houses a Tietz 4K x 4K CCD detector. To support interactive telemicroscopy, high-quality hight-definition television video from the instrument is streamed over the Internet.

TEMs
JEOL-1200 (120 kV) TEM , JEOL-1200 MKII (120 kV) TEM, and JEOL-2000EX (200 kV) cryoelectron microscope.

Light Microscopes
Bio-Rad RTS2000 real-time confocal/multiphoton microscope: An inverted microscope equipped with high-precision XYZ stages under automated computer control for producing of very large field 3-D mosaics. Capable of collecting four-channel images at a speed of up to 120 frames per second. It is also equipped with a Total Internal Reflection Fluorescent Microscope for high sensitive imaging.

Custom Nikon RCM8000 real-timecConfocal/multi-photon microscope: An upright scope featuring high-speed multi-photon capabilities the RTS.

Bio-Rad Radiance 2000 confocal/2-photon microscope: Ultrasensitive upright microscope able to acquire 16-bit data in 1024 x 1024 format using gallium arsenide phosphide PMTs.

Bio-Rad MRC1024 confocal: Inverted microscope equipped with a high-precision XYZ stage under automated computer control that permits acquisition of very large field 3-D montages.

Olympus FluoView1000: Inverted microscope equipped with a high-precision closed-loop XY stage under automated computer control for producing very large field 3-D mosaics. It is also equipped with an environment chamber for long-time live cell imaging.

Olympus spinning disk confocal microscope: Upright microscope equipped with a high-precision closed-loop XY stage and closed-loop Z control with commercial mosaic acquisition software from MicroBrightField. It is equipped with a high-resolution, high-sensitive CCD camera for high-speed mosaic acquisition.

Olympus BX50WI: Specially equipped for cell filling using a Dage-MTI infrared (IR) CCD camera and a Nikon NARISHIGE micromanipulation and microinjection system.

Other Equipment
Bal-Tec HPM 010 high- pressure freezing machine and freeze substitution devices: Combining these techniques provides the best specimen preparation method for obtaining high resolution in situ data.

Cell filling station equipped for intracellular injection: A Dage-MTI IR CCD camera and a Nikon NARISHIGE micromanipulation and microinjection system.

Software

A variety of software packages for image processing and analysis is maintained by the NCMIR, including the full complement of integrated software for handling the acquisition, processing, analysis, management, and display of image data. In addition to in-house software, commercial packages such as the Analyze software package from Dr. Richard A. Robb at the Mayo Foundation/Clinic), Bitplane Imaris, Neurolucida, and Amira, are available. Other software packages are available for image deblurring and deconvolution, including AutoDeblur from AutoQuant.

Publications

1. Price, D. L., Chow, S. K., MacLean, N. A. B., Hakozaki, H., Peltier, S., Martone, M. E., and Ellisman, M. H., High-resolution large-scale mosaic imaging using multiphoton microscopy to characterize transgenic mouse models of human neurological disorders. Neuroinformatics 4:65–80, 2006.

2. Coggan, J. S., Bartol, T. M., Esquenazi, E., Stiles, J. R., Lamont, S., Martone, M. E., Berg, D. K., Ellisman, M. H., and Sejnowski, T. J., Evidence for ectopic neurotransmission at a neuronal synapse. Science 309:446–451, 2006.

3. Giepmans, B. N., Deerinck, T. J., Smarr, B. L., Jones, Y. Z., and Ellisman, M. H., Correlated light and electron microscopic imaging of multiple endogenous proteins using Quantum dots. Nature Methods 2:743–749, 2005.

4. Bouwer, J. C., Mackey, M. R., Lawrence, A., Deerinck, T. J., Jones, Y. Z., Terada, M., Martone, M. E., Peltier, S., and Ellisman, M. H., Automated most-probable loss tomography of thick selectively stained biological specimens with quantitative measurement of resolution improvement. Journal of Structural Biology 148:297–306, 2004