Until recently, it was not possible, due to technical and methodological limitations, to study the structures of macromolecular complexes in living cells. Cryo-EM studies have been performed on isolated macromolecular complexes and organelles. In contrast cells had to be fixed, stained by heavy metals, and sectioned for imaging in an electron microscope. However, current advances in focused ion beam milling (FIBM) and cryo-electron tomography (cryo-ET) permit imaging of macromolecular complexes in near native state in sections of vitrified cells.
Here we present optimization of FIBM protocol to prepare lamellas from mammalian tissue culture cell lines for cryo-ET. Cells grown on golden electron microscopy holey carbon coated grids with Quantifoil® were blotted and vitrified in liquid ethane using Vitrobot Mark 4 (Thermo Fisher Scientific) to prevent formation of crystalline ice. Subsequently, lamellas were milled using focused beam of gallium ions on dual beam scanning electron microscope Versa 3D (Thermo Fisher Scientific). We strive to prepare lamellas with maximal thickness of 250 nm so that they are suitable for imaging in transmission electron microscope operated at 300 kV. Grids with milled lamellas are then transferred, in cryogenic conditions, to electron microscope Titan Krios (Thermo Fisher Scientific) for cryo-ET of selected objects of interest inside the cell.
This research was supported by ERC Grant Agreement 355855 and EMBO Grant Agreement IG 3041 awarded to Dr. Pavel Plevka. We would like to thank to CEITEC 2020 (LQ1601), with financial support from the Ministry of Education, Youth and Sports of the Czech Republic under National Sustainability Program II and Czech Infrastructure for Integrative Structural Biology (LM2015043 funded by the Ministry of Education, Youth and Sports of the Czech Republic).