Tim Laugks

TL;DR: A procedure, based upon focused ion beam (FIB) milling for the preparation of thin lamellae from vitrified cells grown on electron microscopy (EM) grids, which 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.

TL;DR: This work presents a comprehensive description of a cryo-sample preparation workflow incorporating additional conductive-coating procedures, and discusses optimized FIB milling strategies adapted from material science and each critical step required to produce homogeneously thin, non-charging FIB lamellas.

TL;DR: Here, correlative cryo-fluorescence microscopy is used to navigate large cellular volumes and to localize specific cellular targets and it is shown that the selected targets in frozen-hydrated specimens can be accessed directly by focused ion beam milling.

TL;DR: The development of a cryo-stage allowing for spinning-disk confocal light microscopy at cryogenic temperatures is presented and the incorporation of the new hardware into existing workflows for cellular sample preparation bycryo-FIB is described.

TL;DR: A technique to ‘lift out’ samples of interest from high-pressure-frozen specimens expands applications of cryo-electron tomography to multicellular organisms and tissue, extending the range of applications for in situ structural biology.