A.J. Kievits
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Electron microscopy (EM) allows ultrastructural analysis of biological tissues and cells, but images frequently contain artifacts because biological samples have to undergo rigorous preparation to be resistant to vacuum conditions and electron beam exposure. Knowledge about the appearance of image artifacts and how they arise is crucial for their recognition and mitigation and for proper image interpretation. How artifacts appear depends strongly on the electron detection modality and the imaging conditions. Optical scanning transmission EM (OSTEM) is a detection technique compatible with single-beam and multibeam electron microscopes, in which tissue samples are directly deposited on a scintillator for imaging in transmission mode. Here, we identified several types of artifacts that may occur in single-beam and multibeam OSTEM. These artifacts arise or appear as a result of combining established sample preparation protocols with solid scintillator substrates and optical transmission detection. Artifacts can be effectively mitigated or minimized to ultimately enable high quality large-scale 2D and 3D acquisitions.
Traditionally, 3D reconstructions of tissue and cells are achieved by cutting serial thin sections of resin-embedded samples, mounting them on support grids, and imaging with transmission EM. Today, volume EM includes several complementary techniques, each with different resolutions and field-of-view. For example, in array tomography, serial sections are placed on a solid substrate and imaged with scanning EM. In serial block-face scanning EM, a thin tissue slice is removed by an in situ ultramicrotome, and the exposed tissue block face is imaged. With a focused ion beam in a scanning EM, an even thinner slice can be precisely removed. The expanded toolkit has extended volume EM beyond its original application in neuroscience to a wide range of fields.
Advances in volume EM have largely been made possible by improvements in instrumentation, such as more automated workflows and faster and sensitive detectors. Nevertheless, the limited throughput of EMs remains a major bottleneck, especially for large volume imaging. Recent methodological innovations are, however, making possible the imaging of millimeter-sized samples and small organisms. In transmission EM, the throughput is limited by time-consuming sample grid replacement, stage movements and limited fieldof- view at high magnification. Reel translation systems with transparent tape, faster sample stages, larger camera arrays and advanced beam deflection have solved these bottlenecks and increased throughput…
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Traditionally, 3D reconstructions of tissue and cells are achieved by cutting serial thin sections of resin-embedded samples, mounting them on support grids, and imaging with transmission EM. Today, volume EM includes several complementary techniques, each with different resolutions and field-of-view. For example, in array tomography, serial sections are placed on a solid substrate and imaged with scanning EM. In serial block-face scanning EM, a thin tissue slice is removed by an in situ ultramicrotome, and the exposed tissue block face is imaged. With a focused ion beam in a scanning EM, an even thinner slice can be precisely removed. The expanded toolkit has extended volume EM beyond its original application in neuroscience to a wide range of fields.
Advances in volume EM have largely been made possible by improvements in instrumentation, such as more automated workflows and faster and sensitive detectors. Nevertheless, the limited throughput of EMs remains a major bottleneck, especially for large volume imaging. Recent methodological innovations are, however, making possible the imaging of millimeter-sized samples and small organisms. In transmission EM, the throughput is limited by time-consuming sample grid replacement, stage movements and limited fieldof- view at high magnification. Reel translation systems with transparent tape, faster sample stages, larger camera arrays and advanced beam deflection have solved these bottlenecks and increased throughput…
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.
FAST-EM array tomography:
A workflow for multibeam volume electron microscopy
Need for Speed
Imaging Biological Ultrastructure with the 64-beams FAST-EM
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.