Cryoelectron Tomography (CryoET) Centers
The National Network for CryoET was established as part of the Transformative High Resolution Cryo-Electron Microscopy Program in 2020, funding four centers to provide the biomedical research community access to advanced instrumentation for cryoET, cryoET specimen preparation, and collection of high-resolution cryoET data as well as cross-training in cryoET methods.
The network comprises a HUB, The Midwest Center for Cryo-Electron Tomography (MCCET) that performs high-resolution data collection and three “spokes”, or centers that provide optimal sample preparation and screening. The spokes are located at CU Boulder Center for Cryo-ET (CCET), The National Center for In-situ Tomographic Ultramicroscopy (NCITU), and Stanford-SLAC CryoET Specimen Preparation Service Center (SCSC). All of the centers are accessible through competitive peer review of user proposals administered by the HUB and there are no geographical restrictions.
Learn more and apply for access at www.cryoetportal.org
Network Hub: The Midwest Center for Cryo-Electron Tomography (MCCET)
Located at the University of Wisconsin-Madison, MCCET provides access and training in all aspects of the CryoET pipeline and is responsible for the coordination of the national CryoET network activities between the NIH, network centers, and users.
Types of access:
- Routine and advanced cryo-electron tomography (cryo-ET) specimen preparation, data collection, and computation
- Hands-on, remote, and virtual training in cryo-ET specimen preparation, data collection, and data processing and validation
- Academic and commercial data collection services
Instrumentation:
- 300kV Titan Krios G3i Cryo-TEM with Volta phase plates, STEM detector, Falcon 3C direct electron detector, Gatan Bioquantum Post-column Energy Filter with K3 direct electron detector, Ceta CMOS camera.
- 300kV Titan Krios G4i Cryo-TEM with Selectris Energy Filter and Falcon 4i direct electron detector.
- 200kV Talos Arctica with Volta phase plates, STEM detector, Falcon 3C direct electron detector, Gatan Bioquantum Post-column Energy Filter with K3 direct electron detector, Ceta CMOS camera.
- 120kV Talos L120C with Ceta CMOS camera, room temperature specimen holders, Gatan 626 cryotransfer system and holders.
- Aquilos 2 Cryo-FIB-SEM (MCCET) with dedicated cryo-stage cooling, a loading and transfer system, field emission SEM column. Integrated fluorescence imaging (iFLM) for correlative light microscopy.
- Aquilos 2 Cryo-FIB-SEM (CEMRC) with dedicated cryo-stage cooling, a loading and transfer system, field emission SEM column without iFLM.
Fluorescent Light Microscopes:
- Leica DMi8 widefield fluorescence microscope with Alveole PRIMO Micropatterning system.
- Leica M165 Stereo microscope.
- Leica Cryo-CLEM System with cryo-CLEM stage and transfer shuttle, Leica DM6 FS automated wide-field upright microscope and motorized stage; filter systems for DAPI, GFP, Texas Red (mCherry), YFP, CFP, Cy3, and Cy5. Software for automation and image processing.
- Leica Stellaris Five Cryo Confocal microscope.
Sample Preparation:
- FEI Vitrobot Mark IV cryo-plunge freezing system.
- Leica EM GP2 Cryo-plunge freezing system.
- Leica ACE600 System for glow discharge, carbon evaporation, and metal coating.
- Leica EM ICE High pressure freezing system.
- Leica Ultracut Cryo-Ultramicrotome.
- Cryo-grid storage with puck holders
- Dedicated wet and dry lab research spaces for sample handling.
- Stereo microscopes
- Shaking incubators, CO2 incubators
- Biosafety cabinets
CU Boulder Center for Cryo-ET (CCET)
Located at the University of Colorado, CCET will focus on cryo-specimen preparation and cryo-electron tomography (cryo-ET) applied to cells, organelles and large supramolecular assemblies.
Types of access:
- HPF sample preparation
- VitroJet sample preparation
- FIB-SEM sample preparation
- THUNDER-CLEM sample correlation
- Time resolved sample preparation
- Sample screening
- Embedded User Training
Instrumentation:
- Aquilos-2 CryoFIB
- Thunder CLEMVitrobot Mark V
- Cryosol Vitrojet
- Wohlwend High Pressure Freezer
- Tecnai F30; K2; OneView
- Tecnai F20; K3
- GPU work stations
The National Center for In-situ Tomographic Ultramicroscopy (NCITU)
Located at the New York Structural Biology Center, NCITU will be dedicated to developing and providing access to both standard and advanced methods for in-situ cryoET specimen preparation, and to disseminating this expertise through a cryoET cross-training program designed to serve a wide variety of skill levels and career goals.
Types of access:
- HPF sample preparation
- FIB-SEM sample preparation
- cryo-CLEM sample correlation
- Sample screening
- User Training
Instrumentation:
- LeicaDMi8 inverted light microscope, equipped with 4x, 10x and 20x objectives and automation software
- PRIMO micropatterning for fine-tuning cell adhesion to mimic in vivo phenotypes, study cellular mechanisms or isolate cells in reproducible conditions for standardized assays. Cells can be accurately positioned on a EM grid.
- Zeiss LSM900/Airyscan2 confocal microscope with Linkam cryo stage. Up to three standard cryoEM grids can be imaged in a single loading of Linakam cryo-stage cassette. ZenConnect software allows integration of data from FIB-SEM and TEM instruments
- Leica EM-GP2 plunge-freezer. Provides single-sided blotting leaving the specimen unperturbed.
- Aquilos 2 Cryo-FIB DualBeam dedicated to the automated preparation of thin, electron-transparent lamellas for high-resolution cryo-electron tomography.
- GPU workstation for data analysis.
Stanford-SLAC CryoET Specimen Preparation Service Center (SCSC)
Located at Stanford University, SCSC will enable external users to streamline the preparation of samples through providing access to, and support for, advanced cryo-specimen preparation techniques (cryoFLM, cryoFIB, and cryoEM/ET) for a wide range of samples with biomedical applications. The Center will offer a comprehensive program to train and cross-train scientists, regardless of their institutional affiliations, to become independent cryoET investigators.
Type of Access
- Single and Multiple instrument access for routine and advanced cryo-electron microscopy (cryo-ET) specimen preparation
- User training in all aspects of cryo-ET sample preparation and instrument operation
- Full-service support for untrained users
- Academic and commercial user services and training
- User projects with Biosafety Level 1 and 2 specimens
Instrumentation (cryoFIB-SEM and TEM)
- Aquilos2 Cryo-FIB-SEM with field emission SEM column, secondary and backscattered electron detectors, cryo-liftout nanomanipulation and integrated fluorescence imaging (iFLM) for correlative light microscopy.
- Hydra Bio cryo-Plasma-Focused Ion Beam SEM (PFIB-SEM) with secondary (SE), backscattered (BSE) and Scanning Transmission (STEM) electron detectors, cryo-liftout (CLO) nanomanipulation and integrated fluorescence imaging (iFLM) for correlative light microscopy.
- ThermoScientific ‘Tundra’ 100kV cryo-TEM
- ThermoScientific ‘Krios G3i’ 300kV cryo-TEM with Gatan K3 camera, Bioquantum imaging filter (GIF) and Volta phase plate.
Fluorescence Light Microscopes
- Leica DMi8 widefield fluorescence microscope with Alveole PRIMO Micropatterning system.
- Zeiss Airyscan2 (LSM800) confocal with Linkam cryostage
- Zeiss AxioVert inverted brightfield and fluorescence microscope
- Zeiss AxioZoom V16 brightfield and fluorescence stereomicroscope
Sample Preparation
- Leica EM GP2 grid vitrification
- ThermoScientific Mark IV grid vitrification
- Pelco EasiGlow glow discharging of grids
- Leica EM ICE High Pressure Freezing
- Waffle grid preparation
- Leica ACE600 carbon evaporation and metal coating, including Pt, Ir, Au/Pd and Au.
- PRIMO Grid Micropatterning
- Leica VT1200s Vibratome tissue slicing
- Aquilos cryoFIB-SEM lamellae preparation
- Hydra Bio cryo-PFIB-SEM Volume EM imaging
- Cryo-CLEM sample correlation
- Grid screening using a 300kV Titan Krios G3 or 100kV Tundra cryo-TEM
- Zeiss Axiovert for cell culture evaluation
- Zeiss AxioZoom stereo microscope for manipulating fluorescent tissue and organisms
- Biosafety cabinets
- Mammalian cell culture incubators with CO2 and temperature control
- Shaking incubators for microbial cell culturing
- Dedicated wet and dry lab research space for sample handling
- Humidity controlled areas for vitrification activities
- Inventoried cryogenic grid storage using a SubAngstrom puck system