Philipp Sebastian Erdmann

Understanding cellular events in three dimensions (3D) is of great importance for the annotation and illustration of biological processes in a contextual way. Imaging techniques based on electron microscopy (EM), such as those derived from scanning electron microscopy (SEM) and transmission electron microscopy (TEM), provide various options to visualize biological samples at scales ranging from cells to macromolecules in situ . Recently, a series of cryogenic techniques has brought EM‐based imaging to a new level, enabling specimens to retain their hydrated state throughout the sample preparation and imaging steps, thereby offering a near‐native visualization of cellular events. The application of dual‐beam focused ion beam (FIB)‐SEM to biological samples has enabled high‐resolution reconstructions in 3D and streamlined sample preparation workflows for downstream cryo‐electron tomography (cryo‐ET) imaging. However, applications of these technologies to plant materials are limited due to intrinsic characteristics of plant cells (e.g., non‐adhesive growth, large size with a central vacuole, and the presence of cell walls). For the timely application of dual‐beam FIB‐SEM in three‐dimensional subcellular imaging of plant materials, we have recently tested and developed three major workflows with proof‐of‐concept evidence using developing anthers and in vitro ‐cultured pollen tubes based on Aquilos 2 Cryo‐FIB, including (1) room‐temperature FIB‐SEM volume imaging, (2) cryo‐lamellae preparation from cell suspension culture or high‐pressure‐frozen organs for cryo‐ET imaging, and (3) cryo‐FIB‐SEM volume imaging, which will facilitate structural studies of plant materials and provide technical guidance for the broader plant cell biology research community.

Versatile DNA and polypeptide‐based structures have been designed based on complementary modules. However, polypeptides can also form higher oligomeric states. We investigated the introduction of tetrameric modules as a substitute for coiled‐coil dimerization units used in previous modular nanostructures. Tetramerizing helical bundles can run in parallel or antiparallel orientation, expanding the number of topological solutions for modular nanostructures. Furthermore, this strategy facilitates the construction of nanostructures from two identical polypeptide chains. Importantly, tetrameric modules substantially stabilized protein nanostructures against air–water interface denaturation, enabling the determination of the first cryo‐electron microscopy three‐dimensional structure of a coiled‐coil‐based nanostructure, confirming the designed agreement of the modules forming a tetrahedral cage.

Versatile DNA and polypeptide‐based structures have been designed based on complementary modules. However, polypeptides can also form higher oligomeric states. We investigated the introduction of tetrameric modules as a substitute for coiled‐coil dimerization units used in previous modular nanostructures. Tetramerizing helical bundles can run in parallel or antiparallel orientation, expanding the number of topological solutions for modular nanostructures. Furthermore, this strategy facilitates the construction of nanostructures from two identical polypeptide chains. Importantly, tetrameric modules substantially stabilized protein nanostructures against air–water interface denaturation, enabling the determination of the first cryo‐electron microscopy three‐dimensional structure of a coiled‐coil‐based nanostructure, confirming the designed agreement of the modules forming a tetrahedral cage.

Cryo-electron tomography (cryo-ET) and subtomogram averaging (STA) are becoming the preferred methodologies for investigating subcellular and macromolecular structures in native or near-native environments. Although cryo-ET is amenable to a wide range of biological problems, these problems often have data-processing requirements that need to be individually optimized, precluding the notion of a one-size-fits-all processing pipeline. Cryo-ET data processing is also becoming progressively more complex due to the increasing number of packages for each processing step. Though each package has its strengths and weaknesses, independent development and different data formats make them difficult to interface with one another. TOMOMAN (TOMOgram MANager) is an extensible package for streamlining the interoperability of packages, enabling users to develop project-specific processing workflows. TOMOMAN does this by maintaining an internal metadata format and wrapping external packages to manage and perform preprocessing, from raw tilt-series data to reconstructed tomograms. TOMOMAN can also export these metadata between various STA packages. TOMOMAN includes tools for archiving projects to data repositories, allowing subsequent users to download TOMOMAN projects and directly resume processing. By tracking essential metadata, TOMOMAN streamlines data sharing, which improves the reproducibility of published results, reduces computational costs by minimizing reprocessing, and enables the distribution of cryo-ET projects between multiple groups and institutions. TOMOMAN provides a way for users to test different software packages in order to develop processing workflows that meet the specific needs of their biological questions and to distribute their results to the broader scientific community.

Cryo-focused ion beam milling has substantially advanced our understanding of molecular processes by opening windows into cells. However, applying this technique to complex samples, such as tissues, has presented considerable technical challenges. Here we introduce an innovative adaptation of the cryo-lift-out technique, serialized on-grid lift-in sectioning for tomography (SOLIST), addressing these limitations. SOLIST enhances throughput, minimizes ice contamination and improves sample stability for cryo-electron tomography. It thereby facilitates the high-resolution imaging of a wide range of specimens. We illustrate these advantages on reconstituted liquid–liquid phase-separated droplets, brain organoids and native tissues from the mouse brain, liver and heart. With SOLIST, cellular processes can now be investigated at molecular resolution directly in native tissue. Furthermore, our method has a throughput high enough to render cryo-lift-out a competitive tool for structural biology. This opens new avenues for unprecedented insights into cellular function and structure in health and disease, a ‘biopsy at the nanoscale’.

Cryo-electron tomography (cryo-ET) and subtomogram averaging (STA) are becoming the preferred methodologies for investigating subcellular and macromolecular structures in native or near-native environments. While cryo-ET is amenable to a wide range of biological problems, these problems often have data processing requirements that need to be individually optimized, precluding the notion of a one-size-fits-all processing pipeline. Cryo-ET data processing is also becoming progressively more complex due to an increasing number of packages for each processing step. Though each package has its own strengths and weaknesses, independent development and different data formats makes them difficult to interface with one another. TOMOMAN (TOMOgram MANager) is an extensible package for streamlining the interoperability of packages, enabling users to develop project-specific processing workflows. TOMOMAN does this by maintaining an internal metadata format and wrapping external packages to manage and perform preprocessing, from raw tilt-series data to reconstructed tomograms. TOMOMAN can also export this metadata between various STA packages. TOMOMAN also includes tools for archiving projects to data repositories; allowing subsequent users to download TOMOMAN projects and directly resume processing where it was previously left off. By tracking essential metadata, TOMOMAN streamlines data sharing, which improves reproducibility of published results, reduces computational costs by minimizing reprocessing, and enables distributed cryo-ET projects between multiple groups and institutions. TOMOMAN provides a way for users to test different software packages to develop processing workflows that meet the specific needs of their biological questions and to distribute their results with the broader scientific community.

Cryo-focused ion beam milling has enabled groundbreaking structural discoveries in native cells. Progress toward medically relevant applications, however, has been slow. We here present an adaptation of the cryo-lift out procedure for Serialized On-grid Lift-In Sectioning for Tomography (SOLIST), which increases throughput, reduces ice contamination, and enhances sample stability. With these improvements, new specimens, ranging from high-pressure frozen reconstituted LLPS droplets to human forebrain organoids, are accessible to cryo-electron tomography.