Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography

TLDR

Cryoelectron tomography reveals near‑native macromolecular organization but is limited to samples thinner than 0.5–1 µm, so only small prokaryotic cells or peripheral eukaryotic regions can be examined, and cryosectioning introduces artefacts such as compression. The authors describe a focused ion beam milling procedure to prepare thin lamellae from vitrified cells on EM grids. The method employs FIB milling to produce 200–500 nm lamellae from vitrified cells grown on EM grids. The resulting self‑supporting lamellae are free of distortions and enable tomographic studies of any chosen cell region, as demonstrated by a high‑resolution nuclear pore complex structure from a single tomogram.

Abstract

Cryoelectron tomography provides unprecedented insights into the macromolecular and supramolecular organization of cells in a close-to-living state. However because of the limited thickness range (< 0.5–1 μm) that is accessible with today’s intermediate voltage electron microscopes only small prokaryotic cells or peripheral regions of eukaryotic cells can be examined directly. Key to overcoming this limitation is the ability to prepare sufficiently thin samples. Cryosectioning can be used to prepare thin enough sections but suffers from severe artefacts, such as substantial compression. Here we describe a procedure, based upon focused ion beam (FIB) milling for the preparation of thin (200–500 nm) lamellae from vitrified cells grown on electron microscopy (EM) grids. The self-supporting lamellae are apparently free of distortions or other artefacts and open up large windows into the cell’s interior allowing tomographic studies to be performed on any chosen part of the cell. We illustrate the quality of sample preservation with a structure of the nuclear pore complex obtained from a single tomogram.