by Alexander Rigort

Many cells are too thick to study intact in cryo electron tomography. Before the interior of a frozenhydrated cell can be imaged, it must be thinned to electron transparency while maintaining cryogenic conditions. In order to do so, a Aquilos Cryo DualBeam™ cryo focused-ion-beam (cryo-FIB) microscope is used. Utilizing the ion beam of the cryo-FIB microscope, frozen specimens are thinned down to the appropriate thickness of 200-300 nm. Cryo-FIB milling [1-4] opens relatively large and distortion-free windows into the cell’s interior, enabling targeting of structural features within the cellular context. Using a so called in-situ lamella milling or on-the-grid thinning strategy allows directly targeting vitrified cells on EM grids with the ion beam. Thereby, multiple regions of interest, containing highquality electron cryo-lamellas, can be created straight on EM grids. These grids, harboring cryolamellas, are then transferred to a Titan Krios™ transmission electron microscope for high-resolution imaging by cryo electron tomography. Here I will discuss recent advances in cryo-FIB instrumentation and show how newly developed hardware and workflows facilitate cryo sample preparation.

References:
[1] Charting Molecular Landscapes Using Cryo-Electron Tomography. Burbaum et al., Microscopy Today 2017; 25. 26-31.
[2] Cryo-focused-ion-beam applications in structural biology. Rigort & Plitzko, Arch Biochem Biophys. 2015; 581:122-30.
[3] Visualizing the molecular sociology at the HeLa cell nuclear periphery. Mahamid et al., Science 2016 Feb 26;351(6276):969-72.
[4] Optimized cryo-focused ion beam sample preparation aimed at in situ structural studies of membrane proteins. Schaffer et al. J Struct Biol. 2017 Feb;197(2):73-82.