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Publications in Correlative by NOMIS researchers

NOMIS Researcher(s)

Published in

March 1, 2020

Cryo-electron tomography (cryoET) has become a powerful technique at the interface of structural biology and cell biology, due to its unique ability for imaging cells in their native state and determining structures of macromolecular complexes in their cellular context. A limitation of cryoET is its restriction to relatively thin samples. Sample thinning by cryo-focused ion beam (cryoFIB) milling has significantly expanded the range of samples that can be analyzed by cryoET. Unfortunately, cryoFIB milling is low-throughput, time-consuming and manual. Here, we report a method for fully automated sequential cryoFIB preparation of high-quality lamellae, including rough milling and polishing. We reproducibly applied this method to eukaryotic and bacterial model organisms, and show that the resulting lamellae are suitable for cryoET imaging and subtomogram averaging. Since our method reduces the time required for lamella preparation and minimizes the need for user input, we envision the technique will render previously inaccessible projects feasible.

Research field(s)
Health Sciences, Biomedical Research, Developmental Biology

NOMIS Researcher(s)

November 1, 2019

The power of cryo-electron tomography (cryoET) lies in its capability to characterize macromolecules in their cellular context. Structure determination by cryoET, however, is time-consuming compared to single particle approaches. A recent study reported significant acceleration of data acquisition by a fast-incremental single-exposure (FISE) tilt series scheme. Here we improved the method and evaluated its efficiency and performance. We show that (1) FISE combined with the latest generation of direct electron detectors speeds up collection considerably, (2) previous generation (pre-2017) double-tilt axis Titan Krios holders are also suitable for FISE data acquisition, (3) x, y and z-specimen shifts can be compensated for, and (4) FISE tilt series data can generate averages of sub-nanometer resolution. These advances will allow for a widespread adoption of cryoET for high-throughput in situ studies and high-resolution structure determination across different biological research disciplines.

Research field(s)
Health Sciences, Biomedical Research, Biophysics