Parallel cryo electron tomography on in situ lamellae

Abstract

In situ cryo electron tomography of cryo focused ion beam milled samples emerged in recent years as a powerful technique for structural studies of macromolecular complexes in their native cellular environment. The lamella-shaped samples, however, have a limited area and are created with a necessary pretilt. This severely limits the possibilities for recording tomographic tilt series in a high-throughput manner. Here, we utilise a geometrical sample model and optical image shift to record tens of tilt series in parallel, thereby saving time and gaining sample areas conventionally used for tracking of specimen movement. The parallel cryo electron tomography (PACE-tomo) method achieves a throughput faster than 5 min per tilt series and allows the collection of sample areas that were previously unreachable, thus maximising the amount of data from each lamella. Performance testing with ribosomes in vitro and in situ on state-of-the-art and general-purpose microscopes demonstrated the high-throughput and high-quality of PACE-tomo.