Cryo-TEM is increasingly used to determine the structure and ultrastructure of proteins, viruses, bacteria, cells or tissues. Major cryo-TEM acquisition modes are single particle data acquisition, cryo-electron tomography (CET) and micro-electron diffraction (mED). However, cryo-TEM imaging can only be applied for samples with a maximal thickness of approx. 300nm. Cells and tissues in CET or crystals for mED often exceed these dimensions. Samples therefore need to be “thinned” and this can be achieved by cryo-focused ion beam milling prior to cryo-TEM. We have established Australia’s first dedicated cryo-FIBSEM at the Monash Ramaciotti Centre for Cryo EM. Our system consists of a Helios G4 UX FIBSEM and a Leica VCT500 cryo-transfer stage with Leica VCM transfer station and cryo-coater Leica ACE600. The workflow starts with cryo-preservation of crystals and cells via cryo-plunging or tissues via high-pressure freezing. We will discuss examples of how we transfer cryo-preserved specimens to the cryo-FIBSEM, apply cryo-milling procedures and prepare specimens for either mED or CET. We have also started to combine this workflow with cryo-fluorescence microscopy to determine regions of interest before cryo-FIB milling and for labeling structures in cryo-tomograms.
In summary, our workflows will demonstrate how to get high resolution information of highly complex biological systems through a combination of cryo-freezing, cryo-FIB preparation and CET.