The UCSD/NCMIR technological environment houses highly advanced state-of-the-art imaging equipment that is specifically required to accomplish the correlated light and electron microscopy aspects of the project. This technical environment was originally developed to improve staining techniques and subsequent visualization of micro and macro-substructures and is continually broadening these technological capabilities.
The National Center for Microscopy and Imaging Research (NCMIR):
The core laboratory is located on the first floor in the Basic Sciences Building (BSB) of the UCSD School of Medicine. This facility occupies roughly 7500 sq. ft. of space, specifically constructed in 1999-2000 to accommodate the biological, instrument, and computational needs of NCMIR. Resources include four IVEMs, two lower KV electron microscopes, three scanning electron microscopes, ten light microscopes (confocal, multi-photon, and dissection microscopes), as well as molecular biology, histology, and computer resources. There are also facilities for specimen preparation such as vacuum evaporators, high-pressure freezers (Bal-Tec HPM010 and Leica EM PACT2/ RTS), and ultramicrotomes.
Transmission Electron Microscopy:
FEI Titan 80-300 (CTWIN) IVEM/STEM: NCMIR’s centerpiece instrument is a 300 kV STEM and Energy Filter Equipped IVEM manufactured by FEI. The NCMIR Titan is optimized to acquire 3D data sets from specimens up to about 3 mm in thickness. The instrument is also equipped with a microprobe STEM mode that enables sub-1nm resolution to be achieved through 1um thickness of sectioned biological material. With the end-of-column Gatan energy filter (Tridium/GIF), the instrument is ideally suited for Energy Filtered TEM (EFTEM) and Electron Energy Loss Spectroscopy (EELS). The instrument features 5 digital detectors, including two upgraded cameras, based on NCMIR designs, that are custom installed at the end of the energy filter (post-GIF). The first is a DE-12, Direct Detection Device (DDD) detector from Direct Electron, LP (only known installation of this type). The second is an LC-1100, lens-coupled 4K x 4K CCD camera also manufactured by Direct Electron, LP.
JEOL JEM-3200EF: NCMIR also houses a highly customized 300 kV, Energy Filter Equipped IVEM manufactured by Japanese Electron Optics, Ltd. The JEM-3200EF features a high current electron source and Omega-type in-column energy filter. The JEM-3200EF is optimized to acquire 3D data sets from specimens up to about 3 mm in thickness. With the in-column energy filter, the instrument is ideally suited for Energy Filtered TEM (EFTEM) and Electron Energy Loss Spectroscopy (EELS). The instrument features 5 digital detectors, including a DE-12 DDD from Direct Electron, LP and two large format 4K x 4K CCD cameras from Gatan and Tietz Video and Image Processing, respectively.
JEOL JEM-4000EX: NCMIR also houses two JEM-4000EX Intermediate High Voltage (400 kV) Electron Microscopes (IVEM) from Japanese Electron Optics, Ltd.. Both 400 kV instruments feature advanced, high-resolution detection systems. One includes a NCMIR-developed 8k x 8k lens coupled detector that delivers full-resolution (64 Mpixel format) at either 300 or 400 kV. Each JEM-4000EX has been updated with new stage and external computer control systems that allow for automated acquisition of large format tomograms of serial sections. A custom, interchangeable auto-tomography holder allows the automated recording of tomograms from a large series of sections (124+) on a single specially constructed specimen support system.
Scanning Electron Microscopy:
NCMIR currently houses multiple scanning electron microscopes (SEM) dedicated to the process and refinement of ultrawide field, serial blockface scanning electron microscopy (SBFSEM) for large-scale 3D reconstruction from serial sections. Our primary production platform is comprised of a new state-of-the-art, high-speed Zeiss Merlin SEM and a Gatan 3View microtome capable of fully automated collection of SBFSEM data volumes at upwards of 2 TB/week.
The resource currently houses three custom-built 2-photon capable laser-scanning microscopes. The first setup is a highly modified Nikon RCM8000 Laser Scanning Confocal Microscope that features a Nikon Diaphot 300 inverted microscope with epifluorescence and DIC optics. The second 2-photon system is a BIORAD RTS2000 high-speed scanning system, which has been modified to an upright configuration and provides high-resolution images at high speed (video rates). The third is a BioRad Radiance 2000 laser scanning system that has a continuous laser power setting and software for quantifying the intensity of labeling under different specimen preparation conditions. The Resource also features three high performance light microscopes from Olympus, including a high resolution FluoView 1000 confocal microscope, a spinning disk (DSU) confocal system equipped for ultra-wide field mosaicing, and a hybrid FluoView 1000 platform with an integrated spinning disk unit. The latter is ideal for experiments where it is critical to simultaneously acquire with high resolution (FV1000) and high sensitivity (DSU).
NCMIR continues to upgrade and maintain the Keck Center for Integrated Biology, originally established with support from the Keck Foundation in 1999. At this facility, data collected from the instruments are cataloged and transferred to centralized archival storage. Once archived, the data are processed using a myriad of software packages covering the spectrum from image and volume reconstruction to image segmentation to data visualization and analysis. This processing is done on a wide variety of processing systems including laptops and workstations, large memory/CPU servers, and HPC systems, such as SDSC's Triton computational cluster. The resulting data are then catalogued and archived. Cataloguing, archiving, browsing, visualizing and retrieving data is facilitated through a web-based portal to the Cell Centered Database/Cell Image Library. The portal database is effectively cross-referenced with a database used for tracking and managing the hundreds of TBs of raw imaging data and intermediate data products produced. This system facilitates finding data of interest among the TBs available by storing metadata that allows the data to be categorized and filtered according to type and context. High performance networking and data storage equipment further facilitate the processing, replication and management of datasets that are ever increasing in size.
Computation, data storage and network resources for this facility include:
- Two processing nodes with 256 GB RAM and (8) quad-core processors
- Four high end workstations with 24GB-48GB RAM, (6) quad-core processors, and 8TB local storage
- A 30-node computational cluster
- Access and dedicated allocations on High Performance Computing (HPC) resources at the San Diego Supercomputer Center
- Workstations and laptops for research, development and administrative personnel
- 200+ Virtual Machines (VMs) hosted on cost-effective shared Virtual Machine (VM) infrastructure
- Over 1.4 PB of centralized storage
- Two high performance NetApp systems
- one at SDSC for storing Virtual Machine (VM) and database data
- one at NCMIR for storing image data requiring higher availability and high performance
- Access to SDSC's high performance Data Oasis storage cluster, which is directly accessed by their HPC systems
- Access to SDSC's cloud storage
- 10GbE connectivity between 4 separate locations on the UCSD campus
- 10GbE connectivity between data storage and data processing/analysis systems