Volume 1, Issue 1 - Special Issue: Volume Microscopy Across Scales

Tissue slicing is at the core of many approaches to studying biological structures. Among the modern volume electron microscopy (vEM) methods, array tomography (AT) is based on serial ultramicrotomy, section collection onto solid support, imaging via light and/or scanning electron microscopy, and re-assembly of the serial images into a volume for analysis. While AT largely uses standard EM equipment, it provides several advantages, including long-term preservation of the sample and compatibility with multi-scale and multi-modal imaging. Furthermore, the collection of serial ultrathin sections improves axial resolution and provides access for molecular labeling, which is beneficial for light microscopy and immunolabeling, and facilitates correlation with EM. Despite these benefits, AT techniques are underrepresented in imaging facilities and labs, due to their perceived difficulty and lack of training opportunities. Here we point towards novel developments in serial sectioning and image analysis that facilitate the AT pipeline, and solutions to overcome constraints. Because no single vEM technique can serve all needs regarding field of view and resolution, we sketch a decision tree to aid researchers in navigating the plethora of options available. Lastly, we elaborate on the unexplored potential of AT approaches to add valuable insight in diverse biological fields.

Live-cell imaging of fluorescent biosensors has demonstrated that space-time correlations in signalling of cell collectives play an important organisational role in morphogenesis, wound healing, regeneration, and maintaining epithelial homeostasis. Here, we demonstrate how to quantify one such phenomenon, namely apoptosis-induced ERK activity waves in the MCF10A epithelium. We present a protocol that starts from raw time-lapse fluorescence microscopy images and, through a sequence of image manipulations, ends with ARCOS, our computational method to detect and quantify collective signalling. We also describe the same workflow in the interactive napari image viewer to quantify collective phenomena for users without prior programming experience. Our approach can be applied to space-time correlations in cells, cell collectives, or communities of multicellular organisms, in 2D and 3D geometries.

This study explores the efficacy and reliability of high-pressure freezing (HPF) as a sample preparation technique for electron microscopy (EM) analysis across a diverse range of biological samples. Utilizing the HPM Live µ technology, based on the historical hydraulic HPM010 from BalTec, we demonstrate the reliability of our industrial equipment to achieve the critical parameters necessary for vitrification (2076 bars, cooling rate above 2000 K/s). By directly measuring physical values within the HPF chamber, we evaluate the proper functioning of the equipment, contributing to the technique’s reliability. A meticulous approach was adopted for each sample type, acknowledging the uniqueness of each specimen, and associating the final sample analysis with its HPF curve, aiding in protocol optimization. Samples including human cell pellets, cell monolayer, mouse brain and liver biopsies, and Arabidopsis thaliana root and seedlings were processed for EM analysis following HPF. The ultrastructure of each sample type was rigorously examined, revealing homogeneous preservation and minimal ice nucleation artefacts. Overall, our work underscores the robustness and versatility of our HPM Live µ in preserving biological ultrastructure, offering valuable insights for researchers employing EM techniques in diverse biological studies.

Elucidating the 3D nanoscale structure of tissues and cells is essential for understanding the complexity of biological processes. Electron microscopy (EM) offers the resolution needed for reliable interpretation, but the limited throughput of electron microscopes has hindered its ability to effectively image large volumes. We report a workflow for volume EM with FAST-EM, a novel multibeam scanning transmission electron microscope that speeds up acquisition by scanning the sample in parallel with 64 electron beams. FAST-EM makes use of optical detection to separate the signals of the individual beams. The acquisition and 3D reconstruction of ultrastructural data from multiple biological samples is demonstrated. The results show that the workflow is capable of producing large reconstructed volumes with high resolution and contrast to address biological research questions within feasible acquisition time frames.

In this study, we investigate the performance of axial-conical lenses, commonly referred to as Powell lenses, featuring varying fan angles of 5°, 7.5°, 10°, 15°, and 20°. Our objective is to evaluate their suitability for designing a light sheet generator tailored for fluorescence light-sheet microscopy of large samples. Our results indicate that Powell lenses with fan angles of 5° and 7.5° when integrated with additional aspheric components, exhibit optimal characteristics for this application. Specifically, employing a Powell lens with a 7.5° fan angle and 0.2 mm roundness at the tip facilitates the generation of a light sheet ideal for illuminating samples within a size range of 2,000 µm–15,000 µm. To validate the practicality of our optical design for real-world imaging tasks, we conducted imaging experiments on chicken embryos aged between 3 and 7 days. Our light-sheet microscopy system successfully captured intricate structural details, particularly highlighting the ongoing differentiation of the inner anatomy of these specimens. This approach has a high potential to improve the screening of pharmaceutical drugs acting on the vascularization of the chorioallantois membrane (CAM), a technique that is widely used in pharmaceutical research.

Serial block-face (SBF) scanning electron microscopy (SEM) is used for imaging the entire internal ultrastructure of cells, tissue samples or small organisms. Here, we present a workflow for SBF SEM of adherent cells, such as Giardia parasites and HeLa cells, attached to the surface of a plastic culture dish, which preserves the interface between cells and plastic substrate. Cells were embedded in situ on their substrate using silicone microwells and were mounted for cross-sectioning which allowed SBF imaging of large volumes and many cells. A standard sample preparation and embedding protocol for thin section electron microscopy provided already sufficient resolution and image quality to visualize larger structures. To improve resolution and image quality of SBF imaging, we stepwise tested modifications of the protocol, such as the moderate increase of the heavy metal content of the sample. Modifications of the embedding by either the reduction of the resin layer (minimal embedding) or the addition of silver colloid to the resin were evaluated at high and low vacuum imaging conditions. The optimized sample preparation protocol is very similar to the standard preparation protocol for thin section electron microscopy, so that the samples can also be used for this application. The protocol applies a higher concentration of osmium tetroxide, a higher temperature for heavy metal incubation and an additional lead en bloc staining. In summary, the presented workflow provides a generic and adaptable solution for studying adherent cells by SBF SEM.

Freeze substitution is a crucial technique in electron microscopy, enabling the preservation of biological specimen ultrastructure. However, existing tools are primarily designed for 3 mm carriers, limiting their compatibility with larger carriers such as CryoCapsules or 6 mm carriers. In this study, we developed a novel freeze substitution tool, simply named the FS-basket, to address the challenges associated with embedding samples vitrified in larger carriers. The tool comprises a mesh with pore sizes smaller than the sample chunks, deposited below the carrier to maintain a tight seal while allowing for easy fluid exchange.