The Cryo-electron microscopy core facility CEITEC Masaryk University (CEMCOF) provides access to the state-of-the-art electron microscopy instrumentation for cryo-EM room-temperature electron microscopy research in structural and cellular biology by single particles analysis, cryo-electron tomography, and FIB/SEM tomography. As a part of our mission, we support electron microscopy workflows starting from the sample preparation and optimization for the cryo-EM towards advanced sample preparation techniques such as focused ion beam micromachining of cellular lamellas. In addition, the facility continuously adopts emerging techniques in cryo-EM such as electron diffraction tomography (micro-ED) and is available to carry out data analysis. The facility handles BSL1 and BSL2 samples, respectively, and is available to its users for both service and collaborative projects.

Key equipment

• Titan Krios – 300 kV high-end transmission electron microscope aligned for fringe-free imaging (FFI) and equipped with Volta phase plate (VPP), energy filter, and Ametek K3 direct electron camera (Bioquantum K3). The microscope serves for high-edn single particle and cryo-electron tomography data collection.
• Talos Arctica – 200 kV high-end transmission electron microscope equipped with Ceta camera energy filter, and Ametek K2 direct electron detector (Quantum K2). The microscope serves for acquisition of single particle analysis data, high-throughput screening of cryo-EM samples, and acquisition of electron diffraction tomography (microED) data.
• Talos F200C – 200kV scanning transmission electron microscope for operation in both TEM and STEM mode. The microscope is equipped with Falcon 3EC direct electron detector, Ceta-D camera, and Quadro direct electron detection camera. In addition, the microscope is equipped with HAADF detector for STEM imaging and Bruker X-flash detector for EDX mapping. Talos F200C is multi-purpose microscope for life-science and chemistry applications.
• Glacios 2 – 200kV high-end (scanning-)transmission electron microscope for life-science applications, including efficient screening of cryo-EM samples, single particle data acquisition, cryo-electron tomography data acquisition, cryo-STEM imaging and tomography. The microscope is equipped with a Falcon 4i direct electron detector for TEM applications, a Ceta-D camera for electron diffraction tomography, and a set of bright-field, dark-field and high angle annular dark-field detectors (Panther STEM, ThermoScientific) for STEM imaging. Data acquisition can be automated using SerialEM, EPU Multigrid and Tomo5 (including Tomo-Live for real-time tomogram reconstruction). The microscope at CEITEC is the 200th Glacios installed worldwide. The microscope, purchased using resources of the MŠMT program EXCELES, project No. LX22NPO5103, is dedicated to the access proposals submitted exclusively through the National Institute of Virology and Bacteriology.
• Helios V Hydra – dual beam (FIB/SEM) microscope with immersion SEM column for high-resolution SEM imaging. The instrument is equipped with the plasma focused ion beam (four gases – Xe,Ar,O,N) source for efficient milling of biological specimens, cryo-stage, cryo-liftout, and integrated wide-field flourescence microscope. The microscope is used for cryo-FIB lamella preparation and FIB/SEM tomography data acquisition at both room-temperature and cryo-conditions.
• Versa3D – small dual beam (FIB/SEM) equipped with cryo-stage and cryo-FIB/SEM preparation system (Quorum Technologies). The microscope primarily serves for preparation of cellular lamellae by cryo-FIB for cryo-electron tomography.
• Leica CryoCLEM – wide-field flourescence microscope with cryo-stage and 50x cryo-objective (NA 0.9) for correlative light-electron microscopy research on vitrified biological specimens.
• Instrumentation for sample preparation – vitrification robots (ThermoScientific Vitrobot Mark IV, Leica EM GP2) are available for plunge freezing of specimens for cryo-EM. A high-pressure freezer (Leica EM ICE), freeze-substitution unit (Leica EM AFS2), and cryo-ultramicrotome (Leica EM UC7) are available for preparation of cellular or tissue samples for electron microscopy under room-temperature or cryo-conditions.

Expertise

CEMCOF supports services in the the following areas:

• TEM imaging
• SEM imaging
• sample vitrification
• resin embedding
• FIB-SEM tomography
• initial structural screening
• initial cryo-EM screening
• cryo-FIB lamella preparation
• single particle analysis data collection
• cryo-electron tomography data collection
• electron diffraction tomography
• data analysis