Cryo-Focused Ion Beam Milling of Cells

Cryo-electron tomography (cryo-ET) is a robust technique for bringing structural biology into the cellular context. However, transmission electron microscopes (TEM) used for cryo-ET are limited to samples up to 500 nm in thickness, preventing applicability of cryo-ET for most cells. As a result, cellular samples have to be thinned. Traditional EM fixation techniques help overcome this thickness limitation by resin-embedding samples that can be cut into thinner sections. Alternatively, Focused ion beam (FIB) milling is a well-established technique that nanomachines solid-state materials using the FIB without using chemical or mechanical methods. By adapting it to cryogenic conditions, delicate biological materials can be gently and precisely milled to produce thin slices suitable for cryo-ET. Vitrification preserves native structures of biomolecular networks and provides structural stability to frozen cells. Preparing samples using FIB milling of frozen-hydrated cells at cryogenic temperatures is known as cryo-FIB milling, which has emerged as one of the gold-standard techniques for cellular cryo-ET to generate molecular landscapes inside cells. In this chapter, we present a brief history of the field, the fundamentals and practicalities of the technique, and the development of strategies that allow for higher accuracy and efficiency milling before concluding with a discussion of future directions.