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.

Integrated Fluorescence Microscopy (iFLM) for Cryo-FIB-milling and In-situ Cryo-ET.

Correlative cryo-FLM-FIB milling is a powerful sample preparation technique for in situ cryo-ET. However, correlative workflows that incorporate precise targeting remain challenging. Here, we demonstrate the development and use of an integrated Fluorescence Light Microscope (iFLM) module within a cryo-FIB-SEM to enable a coordinate-based two-point 3D correlative workflow. The iFLM guided targeting of regions of interest coupled with an automated milling process of the cryo-FIB-SEM instrument allows for the efficient preparation of 9-12 ∼200 nm thick lamellae within 24 hours. Using regular and montage-cryo-ET data collection schemes, we acquired data from FIB-milled lamellae of HeLa cells to examine cellular ultrastructure. Overall, this workflow facilitates on-the-fly targeting and automated FIB-milling of cryo-preserved cells, bacteria, and possibly high pressure frozen tissue, to produce lamellae for downstream cryo-ET data collection.

Ultrastructural Analysis and Three-Dimensional Reconstruction of Plasmodesmata.

Array tomography (AT) is a new high-throughput imaging method for high-resolution imaging of ultrastructure and for 3-D reconstruction of cells and organelles. Here, we describe the entire procedure for obtaining a spatial image of the distribution of plasmodesmata (PD). As example, the protocol is applied here to reconstruct the number and arrangement of PD between cells undergoing differentiation during Arabidopsis somatic embryogenesis.

Array Tomography Workflow for the Targeted Acquisition of Volume Information using Scanning Electron Microscopy.

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission Electron Microscopy (TEM) provided insight into cell ultrastructure, but in the recent decade, the development of modern Scanning Electron Microscopes (SEM) has changed the way of looking inside the cells. Even though the resolution of TEM is superior when protein-level structural details are needed, SEM-resolution is sufficient for the majority of the organelle-level cell biology-related questions. The advancement in technology enabled automatic volume acquisition solutions such as Serial block-face imaging (SBF-SEM) and Focused ion beam SEM (FIB-SEM). Nevertheless, to this day, these methods remain inefficient when the identification and navigation to areas of interest are crucial. Without the means for precise localization of target areas before imaging, operators need to acquire much more data than they need (in SBF-SEM), or, even worse, prepare many grids and image them all (in TEM). We propose the strategy of "lateral screening" using Array Tomography in SEM, which facilitates the localization of areas of interest, followed by automated imaging of the relevant fraction of the total sample volume. Array tomography samples are conserved during imaging, and they can be arranged into section libraries ready for repeated imaging. Several examples are shown in which lateral screening enables us to analyze structural details that are incredibly challenging to access with any other method.

Clearance by Microglia Depends on Packaging of Phagosomes into a Unique Cellular Compartment.

Phagocytic immune cells such as microglia can engulf and process pathogens and dying cells with high efficiency while still maintaining their dynamic behavior and morphology. Effective intracellular processing of ingested cells is likely to be crucial for microglial function, but the underlying cellular mechanisms are poorly understood. Using both living fish embryos and mammalian macrophages, we show that processing depends on the shrinkage and packaging of phagosomes into a unique cellular compartment, the gastrosome, with distinct molecular and ultra-structural characteristics. Loss of the transporter Slc37a2 blocks phagosomal shrinkage, resulting in the expansion of the gastrosome and the dramatic bloating of the cell. This, in turn, affects the ability of microglia to phagocytose and migrate toward brain injuries. Thus, this work identifies a conserved crucial step in the phagocytic pathway of immune cells and provides a potential entry point for manipulating their behavior in development and disease.