Cryo-focussed ion beam (FIB)-milling can be used to generate thin electron-transparent slices from cells, allowing high-resolution structural studies of the natural cellular environment by in situ cryo-electron tomography (ET). Generating these so called cryo-lamellae is a low-throughput technique and can easily be obstructed by lamella bending, which renders them unusable for subsequent cryo-ET. We hypothesized, that tensions in the sample support are a main cause for lamella bending and improved our protocols by including the milling of “micro-expansion joints” alongside a lamella, which can act as physical buffers to safely absorb tensions. It was demonstrated that adding micro-expansion joints to milling sites drastically increases the milling success rate and that they are fully compatible with the rest of the cryo-ET workflow.
Georg Wolff, Ronald W.A.L. Limpens, Shawn Zheng, Eric J. Snijder, David A. Agard, Abraham J. Koster, Montserrat Bárcena (2019)
“Mind the gap: Micro-expansion joints drastically decrease the bending of FIB-milled cryo-lamellae”, Journal of Structural Biology 208(3)