Martin Koban, Markéta Machálková, Jakub Javůrek
Microscopy and Microanalysis, Volume 29, Issue Supplement_1, 1 August 2023, Pages 1213–1215, https://doi.org/10.1093/micmic/ozad067.624
Published: 22 July 2023
Techniques of volume electron microscopy (EM) have become a strong asset in scientific arsenal when three-dimensional (3D) ultrastructure of biological samples is to be examined. Serial block-face scanning electron microscopy (SBF-SEM) is one of the prominent volume EM methods and has seen a great increase in implementation across research labs, facilities and manufacturers [1]. However, there are still bottlenecks in SBF-SEM associated mainly with complex sample preparation protocols and analysis of vast amounts of generated data [2]. Due to numerous complicated steps, it is usually not possible to execute the entire SBF-SEM experiment using tools from a single vendor and the researchers are forced to laboriously assemble their own customized workflows. Another issue is exchangeability of SBF-SEM systems – some solutions require heavy reconfiguration of the SEM to switch between SBF imaging and standard operation. This makes the microscope a narrowly specialized tool, which is often incompatible with the needs of modern research facilities. Taking these pain points into consideration, we present a lightweight, easy-to-exchange SBF-SEM add-on for TESCAN SEMs, including a dedicated sensitive BSE detector and a software module for volume EM data processing, thus providing tools for smooth execution of complex SBF-SEM experiments.
TESCAN solution for SBF-SEM is based on the integration of in-chamber ultramicrotome Katana by ConnectomX Ltd. [3]. This compact device is installed on a standard SEM stage through a customized adapter which ensures superior mechanical and electrical stability – see Figure 1 (left). The microtome and the adapter can be easily (un)mounted from the stage and replaced with the standard sample holder, an operation that can be performed by a regular microscope user in the order of minutes. This quick installation procedure is particularly beneficial for the systems that are not purely volume EM dedicated tools but should serve a variety of different users, typically in core research facilities.
Another integral part of SBF-SEM experiments is good quality back-scattered electron (BSE) signal detection. Accelerating voltage and electron dose applied during SBF imaging usually have to be minimized as beam penetration must be kept low (to improve resolution in Z) and because resin-embedded samples for SBF-SEM are typically prone to charging and beam damage [2]. At such imaging conditions, detection efficiency of BSE signal is essential to yield images with sufficient signal-to-noise ratio as quickly as possible. Therefore, we have developed a specialized low-energy BSE (LE BSE) detector with improved detection efficiency (particularly at low electron voltages and doses) and modified geometry to allow for imaging at low working distances (thus improving image resolution) – see Figure 1 (right). During SBF imaging, the detector is inserted in between SEM column and Katana microtome with minimum spare gaps to decrease working distance to the sample inside the microtome – see Figure 2 (left). All hardware components of TESCAN SBF-SEM solution have been introduced into 3D model of the SEM chamber, which prevents any potential collision of movable parts – see Figure 2 (right).
Fig. 1. In-chamber Katana microtome mounted on a TESCAN stage (left) and the high-efficiency, low-energy BSE detector dedicated for SBF-SEM application (right).
Fig. 2. Spatial configuration of Katana microtome under SEM column with low-energy BSE detector in between (left). Collision of movable components in the chamber is prevented by TESCAN 3D collision model (right).
Fig. 3. Schematic depiction of an automated SBF-SEM experiment using TESCAN SEM, ConnectomX Katana microtome and open-source acquisition software SBEMimage.
The automated execution of an actual SBF-SEM experiment is ensured by open-source Python-based acquisition software SBEMimage [4]. The application has been accommodated to communicate with TESCAN SEMs and ConnectomX microtome so it can coordinate SEM scanning and block-face cutting. SBEMimage provides users with outstanding flexibility in terms of imaging patterns and conditions, allowing for definition of complex tiled or multi-ROI acquisitions. It also features functions which are especially useful for long experiments, such as automatic SEM focusing, debris detection or remote monitoring with alerts. All SBF-SEM control software can be installed on a single computer to ensure smooth setup of the experiment (see Figure 3).
Acquisition of good quality data is essential in SBF-SEM but unfortunately that is not where the work ends. Multiple processing steps are necessary to transform raw data into scientifically relevant representation, which is usually a 3D volume visualization with subsequent segmentation and quantitative analysis. For these purposes, we have developed a specialized module in TESCAN 3D Analysis Suite (T3D), an in-house software for intuitive and straightforward visualization of volume data – see Figure 4. The software module offers functions optimized for all essential steps of volume EM data processing, from stitching of image tiles, detection and elimination of damaged slices through stack alignment and image enhancement methods to 3D reconstruction and visualization. With these software tools at hand, we are able to cover the entire SBF-SEM workflow in terms of data acquisition and processing (i.e. excluding sample preparation). An example of results achievable with TESCAN SBF-SEM solution can be seen in Figure 5, which showcases 3D analysis of a flatworm and mouse brain samples (data acquired with TESCAN MIRA and CLARA, data processing and visualization in T3D, segmentation in ORS Dragonfly).
By providing integrated and compatible instruments, the presented SBF-SEM solution can help researchers achieve interpretable results faster, eliminating the need to repeatedly switch between different tools or to laboriously develop their own ad hoc workflows. Moreover, easy exchangeability of dedicated hardware components allows for quick reconfiguration of the SEM system, keeping microscope versatility intact. SBF-SEM has already proven its value and importance in biological research and we believe that these developments will make the technique even more accessible to a broad range of scientists [5].
Fig. 4. TESCAN 3D Analysis Suite features intuitive user interface with functions tailored for processing, visualization and analysis of volume EM data.
Fig. 5. 3D reconstructions and segmentation of Macrostomum lignano (flatworm) sample (left) and murine brain sample (right) processed by TESCAN SBF-SEM workflow. Samples courtesy of Dr. P. Ladurner and W. Salvenmoser, Department of Zoology, University of Innsbruck, Austria (flatworm) and Stuart Searle, ConnectomX Ltd. (murine brain).
References
1 D Smith and T Starborg, Tissue and Cell 57 (2019), p. 111. oi:10.1016/j.tice.2018.08.011
2 S Lippens et al. , Methods in Cell Biology 152 (2019), p. 69. doi: 10.1016/bs.mcb.2019.04.002
3 ConnectomX, https://www.connectomx.com (accessed Feb 02, 2023).
4 SBEMimage, https://sbemimage.readthedocs.io (accessed Feb 10, 2023).
5 The authors would like to thank Stuart Searle (ConnectomX Ltd.) and Benjamin Titze (SBEMimage creator and developer) for the collaboration on this project.