Resources

 

FACILITIES:

The core imaging laboratory facilities of the NCMIR are located on the ground floor of the main research building of the School of Medicine, the Biomedical Sciences Building. This facility occupies roughly 7500 sq. ft. of space, specifically constructed in 1999-2000 to accommodate large-scale biological imaging instruments and much of the computational needs of NCMIR. Resources include a fully equipped tissue culture room, a physiology room with microinjection set up, laboratory space for four IVEMs, 2 lower KV electron microscopes, three scanning electron microscopes, ten light microscopes (super-resolution PALM/STORM, lattice light sheet, confocal, multiphoton, 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. More details are below.

Office Space & Administrative Support Facilities: The NCMIR core facility contains office and desk space for Dr. Mark Ellisman (NCMIR Director) and other key resource personnel.  Additional “clean room” space is located on the third floor of BSB for advanced detector engineering and development.  NCMIR also maintains office space at the California Institute for Telecommunications and Information Technology (Calit2) facility, including roughly 3500 sq ft of laboratory space for NCMIR information technology and informatics development.  The Center also maintains roughly 50 sq ft. of dedicated machine room floor space at the San Diego Supercomputer Facility.

Dr. Ellisman is Director of the “Center for Research in Biological Systems” a UCSD research unit that is responsible for the administration of this award and NCMIR support. CRBS maintains administrative personnel for secretarial and fiscal support.

 

RESEARCH RESOURCES:

Specimen Preparation:

The biological research area is furnished with benches, hoods, etc., to prepare biological specimens and conduct biochemical research. The equipment includes pH meters; balances (micro and macro); refrigerators and ultra-cold freezers; an upgraded Kodak Gel Logic 2200 gel and Licor Odyssey visualizer systems for westerns, DNA and protein gels, a tissue chopper, and Vibratome; standard equipment for routine biochemistry, including microfuges, slab gels, tube gels, blots, columns, water baths, a refrigerated bath, homogenizers, a centrifuge (Sorvall), and an ultracentrifuge (Beckman); a Zeiss Microm Cryostat; a liquid-nitrogen-cooled, propane-jet rapid-freezing device; Reichert Ultracut E ultramicrotomes, Reichert-Jung FC4D & FC4E cryokit accessories, and a section counter for serial sectioning; stereotaxic apparatus (David Kopf); electron guns for metal evaporation; high-pressure freezers; a microwave unit with “cold spot” for embedding samples for EM; glass knife breakers (LKB); diamond knives; dissecting microscopes (Nikon, Zeiss, Bausch and Lomb, and A.O. Reichert); upright and inverted conventional light microscopes; and a Vitrobot automated vitrification robot for sample preparation. The micro-iontophoretic, cell-injection station includes electrophysiology equipment (various stimulators and amplifiers), two injection carriers, Narashigi micromanipulators, a light microscope with DIC optics, a glass pipette puller, and a computer-linked cooled CCD camera. An Amaxa nucleoporator is available for more efficient cell transfections.

 

Scanning Electron Microscopy:

NCMIR has three scanning electron microscopes (SEM): (1) a Zeiss Merlin equipped with a Gatan 3View (Denk Slicer) for ultra-widefield serial-blockface scanning electron microscopy (SBEM) for large-scale 3D volume reconstruction, (2) a Zeiss GeminiSEM 300, also equipped with a Gatan 3View for performing SBEM at variable pressure, and a Zeiss AtlasAT system for performing fully automated “extreme-field-of-view” imaging, and (3) a JEOL 5900 Environmental SEM donated from Qualcomm Corp. for general use. Both Zeiss platforms are equipped with high throughput “OnPoint” backscattered electron detectors from Gatan.

 

Transmission Electron Microscopy:

NCMIR houses four Intermediate High Voltage (300-400 kV) Electron Microscopes (IVEMs). Three of these platforms operate at 300 kV, and are specialized for 3D imaging of thick biological specimens, utilizing microscopic tomography techniques, as well as analytical imaging, including energy-filtered TEM (EFTEM) and electron energy loss spectroscopy (EELS).

The FEI Titan 80-300 CTWIN STEM is a STEM and energy-filter-equipped instrument with fully automated capabilities for TEM/STEM tomography (with high angle annular darkfield detection [HAADF]) and EELS. The Titan is equipped with a 4k x 4k (DE-16) Direct Detector, from Direct Electron, LP and multiple 4K x 4K CCD cameras (including a Direct Electron LC-1100 lens couple camera).  The Titan has a pole piece cryo box and is also used for cryoEM, which NCMIR carries out time efficiently by using four cryo specimen holders.  The platform also features an upgraded XFEG high-current FEG source.

In March of 2017, a new cutting edge 300kV IVEM, the FEI Titan Halo, was commissioned into the laboratory to advance technologies for volume-imaging, including multi-tilt serial section EM/STEM tomography and cryo-EM/STEM tomography. This instrument features the higher current XFEG source, XYZ piezo-enhanced compustage, a phase plate, and brightfield and high angle annular darkfield STEM detectors. This new platform is also equipped with the latest (10th) generation 8k x 8k Direct Detector, from Direct Electron, LP.

NCMIR also maintains a third 300-kV platform, a custom JEM-3200EF IVEM from JEOL. This instrument is equipped with an in-column Omega-type energy filter and is the workhorse instrument for the use of EELS to detect new multiple-labeling chemistries (“2-color EM”) with lanthanide elements as well as conduct energy-filtered tomography imaging of thick-stained sections utilizing a Most Probable Loss Tomography strategy developed at NCMIR. Like the Titan 80-300, the JEM-3200 is equipped with a direct detector (DE-12) from Direct Electron. The JEM-3200 is also set up to operate on cryoEM specimens with appropriate anti contaminators and automated low-dose imaging routines.

NCMIR also maintains an older 400-kV IVEM from JEOL (JEM-4000EX). This higher-kV instrument is fielded principally for electron tomography of thick biological specimens. It has a wide-gap pole piece, enabling 360-degree total tilt of specimens, and includes a NCMIR-designed, large-format lens-coupled CCD detector, an 8k x 8k lens-coupled system that delivers greater than 30% contrast at Nyquist and is ideal for ultra-widefield imaging (e.g., whole cell or whole nucleus applications).

The laboratory also has two conventional TEMs, a JEOL JEM-1200 (120-kV) and an FEI Technai 12 (Spirit, 120-kV). The latter is cryoEM-capable and has a 2k Tietz TVIPS camera. Both are useful to investigators with projects involving IVEM access, allowing them to screen thin sections of samples before thicker sections are prepared for examination and recording of tomographic data sets using the higher-kV instruments. We also have four cold stages (three Gatan CTS1100 and one Gatan 626) that can serve either the Titan or the Spirit EM.

 

X-ray Microscopy:

NCMIR has a Zeiss Versa 510 X-ray microscope to perform microCT imaging of small biological samples. The instrument enables precise mapping of regions of interest within specimen volumes prior to performing electron microscopy. The instrument is capable of scanning specimens in a range of 20-180-kV energies and is equipped with a 2k x 2k CCD camera for image collection. The Versa 510 can produce tomographic 3D volumes with ~0.4 um isotropic resolution.

 

Light Microscopy:

NCMIR features a custom-built PALM/dSTORM super-resolution light microscope. This system is equipped with 473-nm, 561-nm, and 671-nm lasers for dye excitation, Acoustic-Optic Tunable Filters (AOTF) to attenuate laser power, and a 405-nm laser for activation, i.e., dEos, dSTORM with 1-MHz modulation capability. For long image acquisitions, the system utilizes an excitation return laser along with a Piezo Z stage to compensate for focus drift. We’ve also equipped the customized platform with two CCD cameras and a beam splitter to image two different dyes. Extending the imaging capabilities of this setup, we’ve also integrated a cylindrical lens to allow for 3D localization microscopy.

NCMIR also features a state-of-the-art, custom Lattice Light Sheet microscope (based on the original design by Eric Betzig).  The LLS instrument is designed for high speed acquisition (100 frames/sec), with high Z resolution (280nm for Structure Illumination Mode), and reduced photo-toxicity.  It is also designed for high sensitivity, with the capability of tracking single molecules.   It is equipped with multiple lasers (multiple excitation) and a high efficiency detector capable of multi-color data acquisition.  Custom modifications include enhancements for improved optical alignment precision and stability and greater laser efficiency.

NCMIR also houses three custom-built, two-photon-capable laser-scanning microscopes. The first is a highly modified Nikon RCM8000 Laser Scanning Confocal Microscope that features a Nikon Eclipse 600FN upright microscope. It has been modified to an upright configuration and provides high-resolution images at high speed (video rates). The second system is a BIORAD RTS2000 high-speed scanning system with epifluorescence and DIC optics. The third is a BioRad Radiance 2000 laser-scanning system equipped with a Nikon E600FN upright microscope that has a direct-detection unit for high-efficiency, two-photon imaging.

NCMIR 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-widefield mosaicking, 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 images with high resolution and high sensitivity. For correlated light and electron microscopy, NCMIR also fields a recently upgraded photooxidation station. Our platform is built upon a Leica SPE II confocal microscope for high-resolution LM imaging before photooxidation. It features a 150W Xenon EPI light source for photooxidation and has a cold stage to keep sample temperature low to preserve ultra structure for EM. A back-illuminated electron-multiplying CCD (EMCCD) adds additional capability for imaging low-fluorescence signal samples, i.e., miniSOG-expressed samples. The progress of photooxidation can now be monitored by this CCD camera with digitally enhanced contrast imaging.

 

Data, Computation, and Networking Resources:

NCMIR maintains an array of custom, commercial, and open-source software for mesoscale image processing and analysis for electron tomography, informatics, and light-microscopy applications. We have a large array of high-performance data storage and computational resources, including access to NSF XSEDE (at SDSC and the Texas Advanced Computing Center [TACC]), commercial cloud services (Amazon and Google), and an assortment of local clusters, including specialized GPGPU cluster resources at the NIH-funded National Biomedical Computation Resource (NBCR, PI - Rommie Amaro). In addition to existing allocation on the Gordon (341 Tflop/s) supercomputer at SDSC, NCMIR benefits from a dedicated allocation of time on SDSC’s newest supercomputer Comet (2 Pflops/s) and high-performance storage in support of our data-management needs. NCMIR’s in-house computing and image-processing facilities consist of 30 large-memory, networked workstations (UNIX/Linux), Macs and PCs, and storage servers supporting roughly 3.5 petabytes of replicated storage, along with optical peripherals for long-term archiving. NCMIR fields multiple video-teleconferencing resources, and large active displays for presentation and meeting/seminar support.