Abstract 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.
Recent advances in electron microscopy techniques have led to a significant scale up in volumetric imaging of biological tissue. The throughput of electron microscopes, however, remains a limiting factor for the volume that can be imaged in high resolution within reasonable time. Faster detection methods will improve throughput. Here, we have characterized and benchmarked a novel detection technique for scanning electron microscopy: optical scanning transmission electron microscopy (OSTEM). A qualitative and quantitative comparison was performed between OSTEM, secondary and backscattered electron detection and annular dark field detection in scanning transmission electron microscopy. Our analysis shows that OSTEM produces images similar to backscattered electron detection in terms of contrast, resolution and signal-to-noise ratio. OSTEM can complement large scale imaging with (scanning) transmission electron microscopy and has the potential to speed up imaging in single-beam scanning electron microscope.
Detailed knowledge of biological structure has been key in understanding biology at several levels of organisation, from organs to cells and proteins. Volume electron microscopy (volume EM) provides high resolution 3D structural information about tissues on the nanometre scale. However, the throughput rate of conventional electron microscopes has limited the volume size and number of samples that can be imaged. Recent improvements in methodology are currently driving a revolution in volume EM, making possible the structural imaging of whole organs and small organisms. In turn, these recent developments in image acquisition have created or stressed bottlenecks in other parts of the pipeline, like sample preparation, image analysis and data management. While the progress in image analysis is stunning due to the advent of automatic segmentation and server-based annotation tools, several challenges remain. Here we discuss recent trends in volume EM, emerging methods for increasing throughput and implications for sample preparation, image analysis and data management.
Journal Article Characterization and Optimization of OSTEM; A Novel Detection Method for Single- and Multi-Beam Scanning Electron Microscopy Get access Arent Kievits, Arent Kievits Department of Imaging Physics, Delft University of Technology – Delft, Netherlands Corresponding author: A.J.Kievits@tudelft.nl Search for other works by this author on: Oxford Academic Google Scholar Job Fermie, Job Fermie Delmic B.V. - Delft, Netherlands Search for other works by this author on: Oxford Academic Google Scholar Peter Duinkerken, Peter Duinkerken Department of Cell Biology, University Medical Centre Groningen- Groningen, Netherlands Search for other works by this author on: Oxford Academic Google Scholar Ryan Lane, Ryan Lane Department of Imaging Physics, Delft University of Technology – Delft, Netherlands Search for other works by this author on: Oxford Academic Google Scholar Elizabeth Carroll, Elizabeth Carroll Department of Imaging Physics, Delft University of Technology – Delft, Netherlands Search for other works by this author on: Oxford Academic Google Scholar Ben Giepmans, Ben Giepmans Department of Cell Biology, University Medical Centre Groningen- Groningen, Netherlands Search for other works by this author on: Oxford Academic Google Scholar Jacob Hoogenboom Jacob Hoogenboom Department of Imaging Physics, Delft University of Technology – Delft, Netherlands Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 28, Issue S1, 1 August 2022, Pages 1458–1460, https://doi.org/10.1017/S143192762200592X Published: 01 August 2022
Journal Article Need for Speed: Imaging Biological Ultrastructure with the 64-beams FAST-EM Get access Arent J Kievits, Arent J Kievits Department of Imaging Physics, Delft University of Technology, Delft, Netherlands Correspondence: A.J.Kievits@tudelft.nl Search for other works by this author on: Oxford Academic Google Scholar B H Peter Duinkerken, B H Peter Duinkerken University Medical Center Groningen, Groningen, Netherlands Search for other works by this author on: Oxford Academic Google Scholar Ben N G Giepmans, Ben N G Giepmans Search for other works by this author on: Oxford Academic Google Scholar Jacob P Hoogenboom Jacob P Hoogenboom Department of Imaging Physics, Delft University of Technology, Delft, Netherlands Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 29, Issue Supplement_1, 1 August 2023, Pages 2105–2106, https://doi.org/10.1093/micmic/ozad067.1091 Published: 22 July 2023
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