Cryo FIB-SEM sample preparation and integrity control

Cryo FIB-SEM sample preparation and integrity control
Contact for more information: 

dr. Jan Andries Post

Sample preparation for Cryo-TEM by Cryo FIB-SEM
Cryo FIB-SEM sample preparation and integrity control

In WP1.2 FEI has developed hardware for Cryo Focused Ion Beam – Scanning Electron Microscope (FIB-SEM). This hardware allows the preparation of samples for cryo tomography using cryo-Transmission Electron Microscopy TEM. The entire workflow from sample preparation to cryo-TEM imaging is shown in the figure above (de Winter et al., J. Struct. Biol. 2010).

In-situ integrity control of a cryo lamella prepared by Cryo-FIB-SEM
Cryo FIB-SEM sample preparation and integrity control

After preparation by cryo FIB milling the cryo lamella  has to be transferred to the cryo-TEM. Once the sample is in the TEM it will become clear whether or not the sample contains the required cells and if the vitreous ice has not become crystalline during the process (quality of the sample). A workflow has been developed to transfer samples from the cryo-FIB-SEM to the cryo-TEM, as shown above. Obviously it would be a great advantage to check the sample quality in-situ in the cryo FIB-SEM, before starting the elaborate transfer process. This has been achieved by cryo Transmission-SEM imaging (Cryo-TSEM) in the FIB-SEM.  The presence or absence of orientation contrast in the Cryo-TSEM indicates the presence or absence of crystalline areas in the sample. Moreover Cryo-TSEM allows visualization of whether or not cells or other structures of interest are present in the lamella (de Winter et al., J. Struct. Biol. 2013). The figure shows the Cryo-TSEM images of crystalline ice. A tilt series of +2, 0 and -2o is shown. A clear change in the contrast between individual grains is shown as a function of the tilt angle, indicating the crystalline nature. This newly developed approach allows halfway quality control of the sample in the workflow shown in the first figure.

15/10/2015