Processes in FCEM
“Processes” are multi-step procedures that require the use of different types of equipment along their value chain.
Cryogenic Electron Microscopy Single Particle Analysis (cryo-EM SPA)
Cryo-EM SPA enables 3D reconstruction of individual proteins and biological complexes with near-atomic resolution by collecting micrographs of purified samples, followed by 2D class averaging and 3D reconstruction.
Description + Tech. Specs. 
Cryogenic Electron Microscopy Single Particle Analysis (cryo-EM SPA)
Head scientist: (soon to be added)
In cryo-EM SPA, purified proteins or protein complexes are suspended in vitreous ice. Currently, the standard technique for vitrification is plunge freezing using semi-automated systems (e.g. Vitrobot, TFS), which preserves the native structure of the protein.
A liquid nitrogen cooled TEM (e.g. 200 kV Glacios or 300 kV Krios, TFS) is then used to semi-automatically collect thousands of movie-stack micrographs to compensate for drift problems. Each stack contains multiple particles randomly oriented within the ice. After aligning the stacks and correcting for the microscope's contrast transfer function, the particles are extracted, the exact orientation of the particles is determined, and the high-resolution 3D structure of the particle is determined.
Class averages indicate different conformational states of the biological complexes present in the sample at the time of vitrification. Cryo-EM SPA is ideally combined with mass spectrometry and molecular dynamics for integrative structural biology analysist.
Sample Preparation
- Semi-Automated Vitrification (Vitrobot, TFS)
- Carbon Coating (208carbon, Cressington)
Data Recording
- 200kV Cryo-TEM (Glacios, TFS)
- 300 kV Cryo-TEM (Krios, TFS)
Data Processing and Simulation
- CryoSparc
- Relion
Cryo-Electron Tomography (cryo-ET)
Cryo-ET allows visualization of proteins and organelles in situ within the cell without isolation and purification, providing structural information about individual proteins as well as their spatial arrangement within the cell.
Description + Tech. Specs.
Cryo-Electron Tomography (cryo-ET)
Head scientist: (soon to be added)
Cryo-ET can provide structural information from complex objects that are too complex and sparse for cryo-EM SPA.
In cryo-ET, a region of interest is imaged in different tilt directions, resulting in a series of 2D images that are aligned and processed into a 3D reconstruction, similar to a CT scan of the human body. Objects of interest are then segmented, can be extracted, and after sub-tomogram averaging, near-atomic resolution can be achieved.
Vitreous samples are prepared by plunge freezing or high pressure freezing. Thin samples can be used immediately for tomography in a 300 kV cryo-TEM. Thicker regions (>500 nm) are thinned and cryo-lamellae are generated in a dual beam scanning electron microscope (FIB/SEM).
In addition, cryo-fluorescence microscopy is often used prior to cryo-lamella generation to identify the locations of fluorescently labeled features of interest.
Sample Preparation
-
Cryo-Lamella Generation (Helios FIB/SEM, TFS) equipped with cryo-Transfer (Leica)
- Plunge Freezing (Vitrobot Mark IV, TFS)
- High-pressure freezing (HPM010, BalTec)
Data Recording
- 300-kV Cryo-TEM (Krios, TFS) equipped with K3 Detektor and Imaging Filter (Gatan)
Data processing and Simulation
- Imod
- CryoCARE
- TomoMemSegTV
- DeePiCt
- Relion
- custom Matlab scripts