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National Center for Microscopy and Imaging Research

Mark H. Ellisman, Ph.D.
P41RR004050
Active

Center Overview

The National Center for Microscopy and Imaging Research (NCMIR) was established to develop computer-aided advanced microscopy for acquisition of structural and functional data in the dimensional range of 1 nm to 100 um, a range encompassing macromolecules, subcellular structures and cells. With novel specimen staining methods, imaging instruments and computational capabilities, researchers are addressing the next great biological challenges in the post-genomic age by situating proteins and macromolecular complexes in their cellular and tissue environments. Resource instruments include intermediate high voltage transmission electron microscopes (IVEMs) and high-speed large-format laser-scanning light microscopes specially modified for "mesoscale" biological microscopy. Our collaboration, service, training and dissemination programs expand the development and use of these technologies to maximize their value to the biomedical community. In this application, NCMIR continues to develop novel methods for imaging biological systems across scales, building upon our successes during the previous funding period and expanding into new areas of technology development. Core technology projects are proposed to develop new staining methods, imaging modes and computational tools for correlated 3D imaging using unique optical and higher voltage electron microscopes. Core research is driven by collaboration with leading scientists in the US and around the world who are addressing key questions in basic cell and neurobiology and conducting translational research in neurodegenerative disease, stroke, cancer, heart failure and infectious disease, to name a few. A unique focus of this resource continues to be the utilization of advanced computational infrastructure to allow remote access to instruments, data and computational resources and to foster collaborations among widely distributed scientists. These new technologies were substantially enhanced over the preceding funding period and will be deployed within our resource to support core and collaborative research. Through this infrastructure, we provide researchers greater access to the Resource through web-based remote control systems for the optical and higher voltage electron microscopes, processing workflows for wide field fluorescence microscopy and electron tomography, and interfaces for computational grids and distributed databases via high-speed networks. In this proposal, we expand these activities to include web-based informatics tools for managing, searching, mining and sharing large multidimensional data sets. These data are shared with the public through the Cell Centered Database, a database for 2D, 3D and 4D microscopy data.

Impact on Human Health

The mission of NCMIR is to develop technologies to bridge understanding of biological systems between the gross anatomical and molecular scales and to make these technologies broadly available to biomedical researchers.

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.