Sven Klumpe

Scanning electron microscopy (SEM) of frozen-hydrated biological samples allows imaging of subcellular structures at the mesoscale in a representation of their native state. Combined with focused ion beam milling (FIB), serial FIB/SEM can be used to build a 3-dimensional model of cells and tissues. The correlation of specific regions of interest with cryo-electron microscopy (cryoEM) can additionally enable subsequent high-resolution analysis. However, the use of serial FIB/SEM imaging-based methods is often limited due to charging artefacts arising from insulating areas of cryogenically preserved samples. Here, we demonstrate the use of interleaved scanning to attenuate these artefacts, allowing the observation of biological features that otherwise would be masked or distorted. We apply our method to samples where inherent features were not visible using conventional scanning. These examples include membrane contact sites within mammalian cells, visualisation of the degradation compartment in the algae E. gracilis and observation of a network of membranes within different types of axons in an adult mouse cortex. The proposed alternative scanning method could also be applied to imaging other non-conductive specimens in SEM.

Cryo-Electron Tomography (cryo-ET) allows to visualize the molecular architecture of pristinely preserved cells and tissues. The workflow of sample preparation for cryo-ET is rather complex; it involves vitrification by rapid freezing followed by cryo-Focused Ion Beam (FIB) milling rendering the volumes of interest thin enough for cryo-ET data acquisition. The established protocols for single cells grown on or deposited on EM-grids are not suitable for multicellular plant tissues. Plunge-freezing does not yield vitrified samples in most cases and must be replaced by high-pressure freezing. This, in turn, necessitates extensive modifications of the subsequent FIB milling procedures. In this communication we describe procedures for sample screening, targeted FIB milling guided by cryo-fluorescence microscopy and a novel lamella trimming step that allows to obtain homogenously thin lamellae suitable for cryo-ET. We have tested all the steps along the workflow with a variety of plant tissues including the moss Physcomitrium patens and tissues of Arabidopsis thaliana and Limonium bicolor. We could demonstrate that the workflow optimized for plant tissues allows to attain subnanometer resolution in cases where subtomogram averaging is applicable.

Focused ion beams (FIB) are widely used instruments in transmission electron microscopy (TEM) sample preparation across scientific disciplines. Generally, site-specific ablation of material is achieved by scanning a highly focused probe across a selected area, leading to the removal of material. However, the geometries of TEM lamellae milled with the FIB are usually highly non-isometric, with their thickness generally being orders of magnitude smaller than their width and length. Here, we explore a changed probe shape for milling. Instead of using a Gaussian-like spot probe ion beam, we characterize the use of the stigmator as quasi-cylindrical lens to create a highly astigmatic beam that we term ‘ion knife’. Using the ion knife allows for material ablation by spreading the current over a larger area and changes the dimension of the probe with an anisotropic change in apparent beam resolution, as observed in spot burn cross-sections. We demonstrate a method to approximate beam shapes by imaging that allows for convenient alignment of parameters for beam shaping. Finally, exploring shaped probes in cryogenic lamella preparation, we demonstrate the feasibility of cellular lamella milling and sectioning of cryo-lift-out volumes with the ion knife.

Although image analysis tools are available within the Galaxy platform, they remain underutilised. During the 2023 BioHackathon Europe, our efforts focused on enhancing the image analysis community in Galaxy by cataloguing and annotating tools and facilitating community discussions to establish naming conventions that promote standardisation. These initial efforts, detailed in the project outcomes, laid the foundation for the ongoing expansion of Galaxy’s image analysis capabilities.Building on these achievements, this year’s work aimed to exploit and demonstrate theGalaxy platform’s full potential to address the needs of the image analysis community.This project involved developing FAIR (Findable, Accessible, Interoperable, and Reusable)image analysis workflows, creating tutorials for the Galaxy Training Network (GTN) to providedocumentation, and fostering broader adoption and facilitating theapplication of these workflows across scientific domains.

Cryo-Electron Tomography (cryo-ET) allows to visualize the molecular architecture of pristinely preserved cells and tissues. The workflow of sample preparation for cryo-ET is rather complex; it involves vitrification by rapid freezing followed by cryo-Focused Ion Beam (FIB) milling rendering the volumes of interest thin enough for cryo-ET data acquisition. The established protocols for single cells grown on or deposited on EM-grids are not suitable for multicellular plant tissues. Plunge-freezing does not yield vitrified samples in most cases and must be replaced by high-pressure freezing. This, in turn, necessitates extensive modifications of the subsequent FIB milling procedures. In this communication we describe procedures for sample screening, targeted FIB milling guided by cryo-fluorescence microscopy and a novel lamella trimming step that allows to obtain homogenously thin lamellae suitable for cryo-ET. We have tested all the steps along the workflow with a variety of plant tissues including the moss Physcomitrium patens and tissues of Arabidopsis thaliana and Limonium bicolor . We could demonstrate that the workflow optimized for plant tissues allows to attain subnanometer resolution in cases where subtomogram averaging is applicable.

Cryo-electron tomography (cryo-ET) has become a key technique for obtaining structures of macromolecular complexes in their native environment, assessing their local organization and describing the molecular sociology of the cell. While microorganisms and adherent mammalian cells are common targets for tomography studies, appropriate sample preparation and data acquisition strategies for larger cellular assemblies such as tissues, organoids or small model organisms have only recently become sufficiently practical to allow for in-depth structural characterization of such samples in situ. These advances include tailored lift-out approaches using focused ion beam (FIB) milling, and improved data acquisition schemes. Consequently, cryo-ET of FIB lamellae from large volume samples can complement ultrastructural analysis with another level of information: molecular anatomy. This review highlights the recent developments towards molecular anatomy studies using cryo-ET, and briefly outlines what can be expected in the near future.