Tomo Live: an on-the-fly reconstruction pipeline to judge data quality for cryo-electron tomography workflows.

Data acquisition and processing for cryo-electron tomography can be a significant bottleneck for users. To simplify and streamline the cryo-ET workflow, Tomo Live, an on-the-fly solution that automates the alignment and reconstruction of tilt-series data, enabling real-time data-quality assessment, has been developed. Through the integration of Tomo Live into the data-acquisition workflow for cryo-ET, motion correction is performed directly after each of the acquired tilt angles. Immediately after the tilt-series acquisition has completed, an unattended tilt-series alignment and reconstruction into a 3D volume is performed. The results are displayed in real time in a dedicated remote web platform that runs on the microscope hardware. Through this web platform, users can review the acquired data (aligned stack and 3D volume) and several quality metrics that are obtained during the alignment and reconstruction process. These quality metrics can be used for fast feedback for subsequent acquisitions to save time. Parameters such as Alignment Accuracy, Deleted Tilts and Tilt Axis Correction Angle are visualized as graphs and can be used as filters to export only the best tomograms (raw data, reconstruction and intermediate data) for further processing. Here, the Tomo Live algorithms and workflow are described and representative results on several biological samples are presented. The Tomo Live workflow is accessible to both expert and non-expert users, making it a valuable tool for the continued advancement of structural biology, cell biology and histology.

Preparing samples from whole cells using focused-ion-beam milling for cryo-electron tomography.

Recent advances have made cryogenic (cryo) electron microscopy a key technique to achieve near-atomic-resolution structures of biochemically isolated macromolecular complexes. Cryo-electron tomography (cryo-ET) can give unprecedented insight into these complexes in the context of their natural environment. However, the application of cryo-ET is limited to samples that are thinner than most cells, thereby considerably reducing its applicability. Cryo-focused-ion-beam (cryo-FIB) milling has been used to carve (micromachining) out 100-250-nm-thin regions (called lamella) in the intact frozen cells. This procedure opens a window into the cells for high-resolution cryo-ET and structure determination of biomolecules in their native environment. Further combination with fluorescence microscopy allows users to determine cells or regions of interest for the targeted fabrication of lamellae and cryo-ET imaging. Here, we describe how to prepare lamellae using a microscope equipped with both FIB and scanning electron microscopy modalities. Such microscopes (Aquilos Cryo-FIB/Scios/Helios or CrossBeam) are routinely referred to as dual-beam microscopes, and they are equipped with a cryo-stage for all operations in cryogenic conditions. The basic principle of the described methodologies is also applicable for other types of dual-beam microscopes equipped with a cryo-stage. We also briefly describe how to integrate fluorescence microscopy data for targeted milling and critical considerations for cryo-ET data acquisition of the lamellae. Users familiar with cryo-electron microscopy who get basic training in dual-beam microscopy can complete the protocol within 2-3 d, allowing for several pause points during the procedure.

A MEMS-based heating holder for the direct imaging of simultaneous in-situ heating and biasing experiments in scanning/transmission electron microscopes.

The introduction of scanning/transmission electron microscopes (S/TEM) with sub-Angstrom resolution as well as fast and sensitive detection solutions support direct observation of dynamic phenomena in-situ at the atomic scale. Thereby, in-situ specimen holders play a crucial role: accurate control of the applied in-situ stimulus on the nanostructure combined with the overall system stability to assure atomic resolution are paramount for a successful in-situ S/TEM experiment. For those reasons, MEMS-based TEM sample holders are becoming one of the preferred choices, also enabling a high precision in measurements of the in-situ parameter for more reproducible data. A newly developed MEMS-based microheater is presented in combination with the new NanoEx™-i/v TEM sample holder. The concept is built on a four-point probe temperature measurement approach allowing active, accurate local temperature control as well as calorimetry. In this paper, it is shown that it provides high temperature stability up to 1,300°C with a peak temperature of 1,500°C (also working accurately in gaseous environments), high temperature measurement accuracy (<4%) and uniform temperature distribution over the heated specimen area (<1%), enabling not only in-situ S/TEM imaging experiments, but also elemental mapping at elevated temperatures using energy-dispersive X-ray spectroscopy (EDS). Moreover, it has the unique capability to enable simultaneous heating and biasing experiments.

A focused ion beam milling and lift-out approach for site-specific preparation of frozen-hydrated lamellas from multicellular organisms.

Cryo-electron tomography provides 3D views of cellular architecture with molecular resolution. A principal limitation of cryo-transmission electron microscopy performed on cells or tissues is the accessible specimen thickness. Recently it has been shown that cryo-focused ion beam milling of plunge-frozen eukaryotic cells can produce homogeneously thin, distortion free lamellas for cryo-electron tomography. Multicellular organisms and tissue cannot be properly vitrified and thinned using this technique because they are considerably thicker. High pressure freezing is therefore necessary to provide optimal preservation. Here, we describe a workflow for preparing lamellas from Caenorhabditis elegans worms using cryo-FIB applied to high pressure frozen samples. We employ cryo-planing followed by correlative cryo-fluorescence microscopy to navigate this large multicellular volume and to localize specific targets within. To produce vitreous lamellas amenable to cryo-TEM observations at these targeted locations, we have developed a dedicated lift-out procedure at cryogenic temperature.