Typically, acutely isolated or cultured cells are seeded on gold or titanium grids with a perforated carbon film (e.g. QuantifoilR) or an SiO2 film (Fig.1, Mahamid et al. 2019). Titanium and SiO2 seem to be stiffer and more stable for the subsequent steps and no additional carbon layer is needed (Toro-Nahuelpan 2019).
Grids are bioactivated by poly-L-Lysin or Fibronectin, trypsinated cells are seeded over night to allow attachment to the carbon layer for the following steps (Mahamid et al., 2019).
For access to cellular samples for successful FIB milling and subsequent analysis in the cryo-TEM, it must be ensured, that the cells are located in or close to the center of the grid squares. Unfortunately, cells tend to grow on grid bars or grow in clusters and are therefore not suitable for FIB milling and analysis with electron transmission. To overcome this challenge, micropatterning allows users to control the location and spreading of cells on the carbon film (Fig. 2), increasing the reliability of the related workflows.
The surface of the grids is coated with polyethylene glycol (PEG) which prevents the attachment of biological material. By removing the coating with a UV laser, the adherence of cells can be specifically controlled and the accessibility for FIB milling and TEM ensured (Toro-Nahuelpan 2019). In addition, specific patterns can be created, thereby influencing the complete cellular architecture and facilitating the investigation of biomechanical phenomena with cryo-electron microscopy.
To keep the samples as close to the native state while being fixed for the electron microscopy, the cells must be very quickly frozen without the creation of destructive ice crystals. This process is called vitrification as the ice becomes amorphous and glass-like (vitreous).
To achieve this for the sample cells, the grids must be rapidly plunged into a suitable cryo-gen (typically ethane, or ethane-propane). In 1981, Jacques Dubochet published the first manual blot-and-plunge method that is still widely used to reach excellent results (Dubochet, J. & McDowall, A. W., 1981).
Before the plunging, excessive liquid must be removed. The standard technique is controlled blotting with filter paper (Fig. 3, Dubochet, J. et al. 1982; Bellare et al., 1988; Frederik, P. M. et al. 1989).
There are different commercial devices available such as the Leica EM GP2 for automatic blotting and plunge freezing. According to the various needs of different sample types, several protocols for sample preparation involving blotting are available (see also here).
After the vitrification the samples must be maintained under cryo-genic conditions throughout the complete workflow. Hence all steps from storage through transfer to the different imaging systems must be performed so that the sample is not devitrified and/or contaminated. This is particularly difficult as such deep cold samples act as a trap for humidity and dust in the close vicinity. Researchers and manufacturers made extensive efforts to develop and provide solutions for keeping the samples safe throughout the different steps of the workflow.
Samples are generally stored in grid boxes in a group of four and the grid boxes are maintained in perforated Falcon tubes in larger