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Mind the gap: micro-expansion joints drastically decrease the bending of FIB-milled cryo-lamellae

By Georg Wolff, Ronald W. A. L. Limpens, Shawn Zheng, Eric J. Snijder, David A. Agard, Abraham J. Koster, Montserrat Bárcena

Posted 31 May 2019
bioRxiv DOI: 10.1101/656447 (published DOI: 10.1016/j.jsb.2019.09.006)

Cryo-focussed ion beam (FIB)-milling of biological samples can be used to generate thin electron-transparent slices from cells grown or deposited on EM grids. These so called cryo-lamellae allow high-resolution structural studies of the natural cellular environment by in situ cryo-electron tomography. However, the cryo-lamella workflow is a low-throughput technique and can easily be hampered by technical issues like the bending of the lamellae during the final cryo-FIB-milling steps. The severity of lamella bending seems to correlate with shrinkage of the EM grid support film at cryogenic temperatures, which could generate tensions that may be transferred onto the thin lamella, leading to its bending and breakage. To protect the lamellae from these forces, we milled "micro-expansion joints" alongside the lamellae, creating gaps in the support that can act as physical buffers to safely absorb material motion. We demonstrate that the presence of such micro-expansion joints drastically decreases lamella bending. Furthermore, we show that this adaptation does not create instabilities that could constrain subsequent parts of the cryo-lamella workflow, as we obtained high-quality Volta phase plate tomograms revealing macromolecules in their natural structural context. The minimal additional effort required to implement micro-expansion joints in the cryo-FIB-milling workflow makes them an easy solution against cryo-lamella bending in any biological sample milled on EM grids.

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