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The technology - CryoET explained
Cryo-electron microscopy (cryo-EM) has emerged to an essential tool in the structural analysis of biological specimen at near atomic resolution. The sample is shock frozen in liquid ethane and embedded in a thin layer of vitreous ice. Consequently, the sample is kept in its native hydrated state and the intrinsic heterogeneity and structure-function relationships are preserved. In order to maintain the specimen in this vitrified state during imaging, the sample is kept at cryogenic temperatures at all time.
Two main approaches to 3 dimensional cryo-EM are single particle analysis (SPA) and cryo-electron tomography (ET). In the former case the three-dimensional structure of the sample is derived by averaging projection images of hundreds of thousands of ideally identical copies of molecules. However, biological assemlies are typically more heterogenious.The use of multiparticle algorythms allow us to extract multiple structures from those specimen.
If multiple copies of the assembly are not available a technique called cryo-electron tomography (cryo-ET) allows us to obtain a 3 dimensional reconstructruction of this uniquly shaped specimen. In cryo-ET this sample is imaged form various angles while being rotated in the electron beam. The resulting tilt-series of projection images can be back projected into a 3D tomogram. Thus, Cryo-ET enables the visualisation of biological assemblies within the cellular context helping to understand it's function within the cell.
Thermo Fisher Scientific Titan Krios G3i
Thermo Fisher Scientific Aquilos 2
Key Features:
- Complete cryo infrastructure including cryo transfer station
- Fully rotatable cryo-stage
- Special Cryo-FIB Autogrids for shallow-angle milling
- Sample shuttle for Autogrids: Cryo-FIB shuttle with integrated shutter system during cryo-transfers
- Sample shuttle compatible with the Leica Confocal SP8 for direct transfer
- In-chamber retractable sputter coater for applying conductive coatings
- GIS-system for applying protective coatings
- MAPS software for correlative workflow
- AutoTEM software for automated lamella preparation
- Kleindiek micromanipulator for liftout
Leica TCS SP8
Key Features:
- Complete cryo Infrastructure including cryo transfer station
- HCX PL APO 50x CLEM cryo-Objektive
- SP8 confocal optical system + SP8 LIGHTNING software
- Compatible with MAPS software for correlative workflow
Laser Lines
405,488,552,638
Detectors
1x PMT detector, 2x HyD detectors
Filters
DAP (Em: 325-375)
N2.1 (Em: 515-561)
I3 (Em: 450-490)
GFP (Em: 450-490)
PRIMO for micropatterning
A micropatterning system that allows control of cell adhesion, spreading and shape on EM grids. It enables precise cell positioning and optimized cell spreading, while leaving the surface of the EM grid undamaged. The software automatically aligns your patterns within the mesh of your EM grid, ensuring that your cells are optimally positioned for further processing.
