Resource Overview

Facilities:

Laboratory: The National Center for Microscopy and Imaging Research (NCMIR) is located within the School of Medicine at the University of California, San Diego. UCSD provides an outstanding environment for the success of this project due to its strong record of high-impact research, spanning campus departments and partnerships with private industry, government, and the broader scientific community. As one of the leading research institutions in the nation, UCSD continues to invest in advanced infrastructure, including high-speed network capabilities and resources for developing and maintaining shared research facilities.

Located on the ground floor of the main research building of UCSD’s School of Medicine, the Biomedical Sciences Building, NCMIR occupies roughly 7500 sq. ft. of space specifically constructed to accommodate largescale 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 three IVEMs, 2 lower KV electron microscopes, three scanning electron microscopes, numerous light microscopes (including lattice light sheet, PALM/dSTORM, 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 HPM-10, Leica EM ICE, Leica EM PACT2/ RTS), and ultramicrotomes.

Office (Administrative Support): The NCMIR core facility contains office and desk space for Dr. Mark Ellisman (NCMIR Director and PI) and other key lab personnel. Additional clean room space is located on the third floor of BSB for advanced instrumentation engineering and development. NCMIR also maintains office space at the Qualcomm Institute, the UCSD division of the California Institute for Telecommunications and Information Technology (Calit2), including roughly 3500 sq. ft. of laboratory space for NCMIR information technology and informatics development. Additional offices and roughly 100 sq. ft. of dedicated machine room floor space is also maintained at the San Diego Supercomputer Center, housing production computing infrastructure and roughly 3.5 PB of replicated storage.

Major Equipment:

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 liquidnitrogen-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 (Leica EM ICE, Leica EMPACT2, Bal-Tec HPM-10); freeze-substitution units (2x Leica AFS2); 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 high-performance scanning electron microscopes (SEM): (1) a Zeiss Merlin (Gemini II) equipped with a Gatan On-Point backscatter detector (BSD) and Zeiss SenseBSD. This is a multi-use platform routinely used for high-throughput, volumetric scanning of serial sections (on silicon wafers) using Zeiss’ AtlasAT (array tomography) software. The platform is also compatible with the Gatan 3View serial blockface (SBEM) ultramicrotome and serves as ‘hot swap’ backup platform for this process. In April 2018, Zeiss upgraded (at their cost) the column of the Merlin Gemini II to an improved configuration for volumetric imaging (at the specification of the GeminiSEM450).

Some usage time on the Merlin is also reserved for research and development into new methods and specialized probes for multi-color SEM. To support this use, we added a new Bruker Quantax QUAD EDS system. This system has a 4x15 mm 2 active area, which is ideal for EDS analysis at very high magnification (or for analysis of highly sensitive samples). Due to its annular design, where the active areas are placed around a hole for the primary beam, each segment absorbs X-ray quanta from a different azimuth angle, allowing a more complete mapping of rough surfaces and particles. The other unique feature of the annular design is that there is no “fixed” analytical working distance, allowing samples to be measured at nearly any working distance. In fact, the design of the QUAD detector allows the active area to get extremely close to the sample surface, giving an extremely high solid angle of up to 1.2 Steradians at about 3mm from the detector.

(2 & 3) NCMIR also operates two production platforms for ultra-widefield SBEM in support of large-scale 3D volume reconstruction. Our newest platform (commissioned in August 2023) is a Zeiss GeminiSEM 450. This modern variable pressure instrument is equipped with a Gatan 3View and OnPoint BSD, a Zeiss SenseBSD, and an Evactron Zephyr plasma cleaner. It is a workhorse platform for SBEM, optimized for resolution, stability and ease-of-use. Our second SBEM platform is a Zeiss GeminiSEM300. Like the 450, it is configured for variable pressure operation and features a Gatan 3View and OnPoint BSD. This instrument is also equipped to perform automated array tomography with an included Zeiss AtlasAT system. Both the GeminiSEM450 and 300 include a NCMIR developed focal charge compensation system to vastly improve the performance of SBEM for nonconductive or charge-prone samples.

Intermediate Voltage Transmission Electron Microscopy (IVEM): NCMIR fields four intermediate high voltage (200-300 kV) electron microscopes (IVEMs). Each 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 ThermoFisher/FEI Titan Halo was procured in 2017 with an NIH HEI grant (1S10OD021784-01A1) to advance technologies for volume imaging, including multi-tilt serial section EM/STEM tomography and cryoEM/STEM tomography. This instrument features a higher current XFEG source, XYZ piezo-enhanced compustage, a Volta phase plate, and FEI’s full suite of bright-field, annular dark-field, and high angle annual dark-field STEM detectors. In 2017, we also equipped this platform with an 8k x 8k direct detector (DE-64) from Direct Electron, LP. The Titan has a polepiece cryo box and is also used for cryoEM, which NCMIR performs using four side-entry cryo-holders, including a specialized Gatan CT3500R tilt-rotate cryo-holder for multi-tilt cryo-tomography (and cryo STEM tomography).

The ThermoFisher/FEI Titan 80-300 CTWIN STEM is a STEM and energy-filter-equipped instrument with fully automated capabilities for TEM/STEM tomography. Like the Titan Halo, this platform is equipped with FEI’s full suite of bright-field, annular dark-field, and high angle annual dark-field STEM detectors. This instrument is also equipped with a Gatan Tridium GIF end-of-column energy filter for EFTEM/ EELS – including most-probable loss tomography (MPL). The Titan is equipped with two direct detectors from Direct Electron, a 4k x 4k DE-16 DD in a pre-GIF configuration (for EM tomography) and a 4k x 5k DE-20 DD in post-GIF configuration (for multicolor EM). The Titan also has a pole piece cryo box and is used for LN temperature cryoEM, which NCMIR performs using four side-entry cryo-holders.

The ThermoFisher/FEI Talos F200C is a STEM and energy-filter-equipped instrument with complete, automated capabilities for energy filter enabled, multiple label transmission electron microscopy (e.g., ColorEM) and high throughput 3D electron microscopic tomography (3D EM) of semi-thick epoxy-embedded samples of cells and tissues (non-Cryo). It is configured to refine and replicate the capabilities of the JEOL JEM-3200EF (described below) at a more accessible specification (i.e., 200keV). Like the Titan Halo and 80- 300, this platform is equipped with FEI’s full suite of bright-field, annular dark-field, and high angle annual dark- field STEM detectors. This instrument is also equipped with a modern Gatan Continuum end-of-column energy filter and DE Apollo Direct Detector, ideal for high-throughput volume imaging. This platform is also equipped with a motorized azimuthal tilt-rotate specimen holder (Fischione Instruments model 2045) for performing automated multi-tilt EM tomography. This instrument was procured in 2023, via an NIH High End Instrumentation grant (S10OD034447).

The JEM-3200EF IVEM from JEOL is equipped with an in-column Omega-type energy filter and a DE-12 DD from Direct Electron. For multiple decades, this instrument has served as the development platform to design, test and troubleshoot new analytical methods for ColorEM and EFTEM strategies (e.g., MPL). This instrument will continue in this capacity, until it is decommissioned due to a lack of serviceability and/or is replaced.

Conventional Transmission Electron Microscopy (TEM): The laboratory also has two conventional TEMs, a JEOL JEM-1200 (120-kV) and a 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 the screening of thin sections of samples before thicker sections are prepared for examination and recording of tomographic data sets using the higher-kV instruments.

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 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 (280 nm for structure illumination mode), and reduced phototoxicity. 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 fields a custom-built PALM/dSTORM super-resolution system. 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, e.g., 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. The customized platform is also equipped 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 fields a custom-built PALM/dSTORM super-resolution system. 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, e.g., 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. The customized platform is also equipped 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 three high-performance light microscopes from Olympus, including a high-resolution FluoView 3000 confocal microscope, a FluoView 1000 confocal system, and a spinning disk (DSU) confocal system equipped for ultra-widefield mosaicking.

The Center also fields a recently upgraded photo-oxidation station. This platform is built upon a Leica SPE II confocal microscope for high-resolution LM imaging before photo-oxidation. It features a LED light source for improved efficiency, repeatability and sensitivity for photo-oxidation and has a cold stage to keep sample temperature low to preserve ultrastructure for EM. A back-illuminated electron-multiplying CCD (EMCCD) adds additional capability for imaging low-fluorescence signal samples (e.g., miniSOG-expressed samples). The progress of photo-oxidation 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. NCMIR’s in-house computing and image-processing facilities consist of ~30 large memory CPU and high-performance GPU enabled servers, desktop/visualization workstations, and storage servers supporting roughly 3.5 petabytes of replicated storage. NCMIR fields multiple video- teleconferencing resources, and large active displays for presentation and meeting/seminar support.

NCMIR also benefits from networked access and dedicated allocations on petascale, high-performance data storage and computational resources, including a dedicated allocation of cycles (1mil/yr) on SDSC’s XSEDE cluster (Expanse), commercial cloud services (NIH Cloud Commons, Amazon Web Services and Google Cloud), and an assortment of research clusters at UCSD. The resource also benefits from dedicated access to specialized, high performance data cache and GPGPU cluster resources associated with the NSF’s National Research Platform (NRP), which are both scalable and optimized to the AI/ML technologies that will be deployed for computational multiscale modeling and data processing (e.g., volume reconstruction and segmentation) serial EMT data.

Protocols

NCMIR protocols are managed and openly disseminated via Protocols.io. Access protocols here.

List of protocols available for download (as of Oct 1., 2023):

• Photo-oxidation using MiniSOG with EM Preparation of Transfected Culture Cells


• Preparation of Biological Tissues for Serial Block Face Scanning Electron Microscopy (SBEM)


• Preparation of Cultured Cells for Serial Block Face Scanning Electron Microscopy (SBEM)


• Correlative Microscopy for Localization of Proteins in Situ: Pre-embedding Immuno-Electron Microscopy Using FluoroNanogold, Gold enhancement, and Low-Temperature Resin


• Ln-DAB2 Solutions


• Lanthanum DAB metals, Ln-DAB2 labeling of APEX2


• Creating Gelvatol Solution


• Karnovsky’s Modified Fixative


• 0.3M Sodium Cacodylate Buffer pH 7.4 Stock Solution Recipe


• Ringer’s Solution

Please email any questions regarding protocols/SOPs to Mason Mackey