• Applied Microscopy
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• v.46 no.2
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• pp.100-104
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• 2016

Electron microscopy is currently the only available technique with a spatial resolution sufficient to identify fine neuronal processes and synaptic structures in densely packed neuropil. For large-scale volume reconstruction of neuronal connectivity, serial block-face scanning electron microscopy allows us to acquire thousands of serial images in an automated fashion and reconstruct neural circuits faster by reducing the alignment task. Here we introduce the whole reconstruction procedure of synaptic network in the rat hippocampal CA1 area and discuss technical issues to be resolved for improving image quality and segmentation. Compared to the serial section transmission electron microscopy, serial block-face scanning electron microscopy produced much reliable three-dimensional data sets and accelerated reconstruction by reducing the need of alignment and distortion adjustment. This approach will generate invaluable information on organizational features of our connectomes as well as diverse neurological disorders caused by synaptic impairments.

• Applied Microscopy
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• v.41 no.3
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• pp.147-154
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• 2011

Backscattered electrons (BSE) are generated at the impact of the primary electron beam on the specimen. BSE imaging provides the compositional contrast to resolve chemical features of sectioned block-face. A focused ion beam (FIB) column can be combined with a field emission scanning electron microscope (FESEM) to ensure a dual (or cross)-beam system (FIB-FESEM). Due to the milling of the specimen material by 10 to 100 nm with the gallium ion beam, FIB-FESEM allows the serial block-face (SBF) imaging of plastic-embedded specimens with high z-axis resolution. After contrast inversion, BSE images are similar to transmitted electron images by transmission electron microscopy. As another means of SBF imaging, a specialized ultramirotome has been incorporated into the specimen chamber of FESEM ($3View^{(R)}$). Internal structures of plastic-embedded specimens can be serially revealed and analyzed by $3View^{(R)}$ with a large field of view to facilitate three-dimensional reconstruction. These two SBF approaches by FESEM can be employed to unravel spatial association of (sub)cellular entities for a comprehensive understanding of complex biological systems.

• Applied Microscopy
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• v.47 no.3
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• pp.171-175
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• 2017

Negative staining has been traditionally used for exosome imaging; however, the technique is limited to surface topology only and can cause staining artifacts. Therefore, to analyze the internal structure of exosomes, we employed a method of block preparation, thin sectioning, and electron tomography. In addition, an automatic serial sectioning technique with 15-nm thickness through focused ion beam was employed to observe the three-dimensional structure of exosomes of various sizes. Cryo-transmission electron microscopy revealed the near-to-native structure of exosomes.

• Applied Microscopy
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• v.46 no.4
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• pp.171-175
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• 2016

A distinctive neuronal network in the brain is believed to make us unique individuals. Electron microscopy is a valuable tool for examining ultrastructural characteristics of neurons, synapses, and subcellular organelles. A recent technological breakthrough in volume electron microscopy allows large-scale circuit reconstruction of the nervous system with unprecedented detail. Serial-section electron microscopy-previously the domain of specialists-became automated with the advent of innovative systems such as the focused ion beam and serial block-face scanning electron microscopes and the automated tape-collecting ultramicrotome. Further advances in microscopic design and instrumentation are also available, which allow the reconstruction of unprecedentedly large volumes of brain tissue at high speed. The recent introduction of correlative light and electron microscopy will help to identify specific neural circuits associated with behavioral characteristics and revolutionize our understanding of how the brain works.

• Applied Microscopy
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• v.40 no.4
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• pp.177-183
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• 2010

A focused ion beam (FIB) system produces a beam of positive ions (usually gallium) which are heavier than electrons and can be focused by electrostatic lenses into a spot on the specimen. With its ability milling of the specimen material by 10 to 100 nm with each pass of the beam, FIB is widely adopted in materials science, semiconductor industry, and ceramics research. Recently, FIB has been increasingly employed in the field of biomedical sciences. Here we provide a brief introduction to FIB and its applications for a wide variety of biomedical research. The surface of specimen can be in situ processed and quasi-real time visualized by two beam combination of FIB and field emission scanning electron microscope (FESEM). Due to its milling process, internal structures can be exposed and analyzed: yeast cells, fungus-inoculated wheat leaf, mannitol particles in inhalation aerosols, and oyster shell. Serial blockface tomography with the system kindles 3-dimensional reconstruction researches in the realm of nervous system and life sciences. Two-beam system of FIB/FESEM is a versatile tool to be utilized in the biomedical sciences, especially in 3-dimensional reconstruction studies.