Tackling ice contamination in cryo-ET

Cryogenic electron tomography (cryo-ET) is an imaging technique that allows the reconstruction of high-resolution models of protein complexes in their near-native cellular environment. In cryo-ET a sample is first vitrified, then thinned to the appropriate thickness (100-300 nm) using a focused ion beam (FIB) inside a scanning electron microscope (SEM), and lastly, a series of images are captured with a transmission electron microscope (TEM). These 2D images are then used to reconstruct a 3D volume, a tomogram. From the tomogram, high resolution structural models of proteins of interest can be reconstructed. During all these steps the sample must remain in a vitrified state at ~ -160 °C.

However, ice contamination of the sample has been one of the biggest challenges hindering researchers from moving further. One of the two major contamination sources is the contamination with ice crystals during transport of the sample. Samples transported or handled in liquid nitrogen can easily be contaminated with ice crystals that have formed when the liquid nitrogen, or the tools that are used in liquid nitrogen, have been exposed to the open air. Samples transported in low vacuum can also attract water molecules that form crystals on the surface of the sample. Ice crystals obscure regions of interest, hampering the acquisition of meaningful tomograms.

The second major contributor to contamination occurs even in the high vacuum of the FIB/SEM chamber. Here, water molecules can deposit and form a crystalline ice layer on the lamellae at a rate of around 50 nm per hour. With the process of lamellae milling lasting many hours, this can cause a thin, ~150 nm lamella to be substantially thicker after all milling is done. The thick crystalline ice layer substantially reduces the quality of the acquired tomogram.

In this webinar, our application specialist Caspar Jonker is going to introduce the crucial steps in the workflow that contribute most to sample ice contamination. We will also dive into the new developments that aim to minimize contamination in cryo-ET altogether. Register now to reserve your spot!