To fully investigate complex biological mechanisms, life science researchers require reliable structural information of target molecules within their subcellular context. To achieve this, the target molecules and their cellular environment need to be accurately resolved at subnanometer resolution.
Leica Microsystems and Thermo Fisher Scientific have collaborated to create a fully integrated Cryo Electron Tomography workflow that responds to these research needs. Safe sample and data transfer between instruments ensure easy navigation to the target cellular regions and reliable results at subnanometer resolution.
Data courtesy of Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany
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The investigated cells are grown on an electron microscopy grid.
Prior to freezing, the sample is maintained in an humidity-controlled environmental chamber at a stable temperature.
The sample is then vitrified by plunge freezing with the automatic plunge freezer EM GP2.
The vitrification process inhibits ice crystal formation ensuring the cellular content is as close to the native state as possible.
For an efficient workflow, suitable cells and target regions have to be preselected. This is achieved by using the cryo light microscope Leica EM Cryo CLEM. The sample can then be transferred to the Thermo Scientific™ Aquilos™ for milling. Full connectivity between the instruments means that the defined region and coordinates are securely and precisely transferred for immediate relocation by the Aquilos. No need to invest time searching for the relevant target positions!
To avoid contamination of the precious specimen, the Integrated Cryo Tomography workflow ensures a secure transfer of the sample between the systems involved. The dedicated cartridge system safeguards your sample throughout the workflow providing the solid fundament for your reliable scientific results.
(Thermo Scientific, Krios and Aquilos are Trademarks by Thermo Fisher Scientific.)
After preselection and targeting in the EM Cryo CLEM, the sample is transferred to the Thermo Scientific Aquilos, a dedicated Cryo DualBeam electron microscope.
In the past, it was impossible to resolve the interior of cells at subnanometer resolution as many samples were simply too thick to be imaged by cryo-electron tomography.
The Aquilos overcomes the thickness limitations by employing a scanning electron beam (SEM) and a focused ion beam (FIB). While the electron beam is used for imaging, a beam of gallium ions ensures the precise milling of vitrified cells.
By the milling process, a thin ice sheet, the on-grid lamella is created and which can then be examined using electron tomography.
The EM grid that carries the cells including the on-grid lamella, is transferred from the Aquilos to the Thermo Scientific Krios™ G3i (Cryo TEM).
The Cryo TEM images the area of interest multiple times from different angles by tilting the sample incrementally.
The single images are computationally aligned and reconstructed to generate a three-dimensional tomogram from the lamella and its content in subnanometer resolution.