Beam deceleration for block-face scanning electron microscopy of embedded biological tissue

Ohta, Keisuke; Sadayama, Shoji; Togo, Akinobu; Higashi, Ryuhei; Tanoue, Ryuichiro; Nakamura, Kei‐ichiro

doi:10.1016/j.micron.2011.11.001

Cited by 98 publications

(85 citation statements)

References 26 publications

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“…Observation of the surface of resin embedded biological samples using backscattered electron imaging of SEM provides an image that is similar to TEM results (19,20 …”

supporting

confidence: 60%

“…Observation of the surface of resin embedded biological samples using backscattered electron imaging of SEM provides an image that is similar to TEM results (19,20). Recently, as an alternative for obtaining TEM serial sectioning images, two serial sectioning techniques, using a microtome or focused ion beam (FIB) integrated inside a SEM, known as serial block-face SEM (SBF-SEM) (20) dimensional image using software from the hundreds of SEM images obtained by repeated sectioning and observation, and have proved to be successful for biological studies, such as following axons and synaptic connections (21), and clarifying mitochondrial fusion and fission (22).…”

supporting

confidence: 57%

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Ring-Mesh Model of Proteoglycan Glycosaminoglycan Chains in Tendon based on Three-dimensional Reconstruction by Focused Ion Beam Scanning Electron Microscopy

Watanabe

Kametani

Koyama

et al. 2016

Journal of Biological Chemistry

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Tendons are composed of collagen fibrils and proteoglycan predominantly consisting of decorin. Decorin is located on the d-band of collagen fibrils, and its glycosaminoglycan (GAG) chains have been observed between collagen fibrils with transmission electron microscopy. GAG chains have been proposed to interact with each other or with collagen fibrils, but its threedimensional organization remains unclear. In this report, we used focused ion beam scanning electron microscopy to examine the three-dimensional organization of the GAG chain in the Achilles tendon of mature rats embedded in epoxy resin after staining with Cupromeronic blue, which specifically stains GAG chains. We used 250 serial back-scattered electron images of longitudinal sections with a 10-nm interval for reconstruction. Three-dimensional images revealed that GAG chains form a ring mesh-like structure with each ring surrounding a collagen fibril at the d-band and fusing with adjacent rings to form the planar network. This ring mesh model of GAG chains suggests that more than two GAG chains may interact with each other around collagen fibrils, which could provide new insights into the roles of GAG chains.Tendon is primarily composed of collagen fibrils and the non-collagen matrix (1). Canty et al. (2) suggested that plasma membrane protrusions called fibripositors contribute to the parallel arrangement of collagen fibrils in tendon, especially in the embryonic stage. Decorin and biglycan belong to the small leucine-rich proteoglycan family and consist of a core protein containing leucine repeats with a covalently attached chondroitin/dermatan sulfate glycosaminoglycan (GAG) 2 chain. The majority (Ͼ90%) of the tendon proteoglycan is decorin, while tendon also contains small amounts of biglycan (3). The decorin core protein binds to the surface of collagen fibrils by non-covalent binding and the attached decorin GAG chain extends from the core protein to associate closely with another decorin GAG chain on an adjacent fibril surface (4 -6).Some studies have suggested that the GAG chain forms bridges between collagen fibrils to guarantee mechanical integrity (4, 7-9). In contrast, other studies have suggested that the GAG chain is irrelevant to mechanical integrity (3, 10 -12), because results of mechanical tests using decorin-knock-out mice vary depending on the type and region of the tendon (13). Some researchers also reported that decorin might restore the alignment of the collagen fibrils deformed under load, rather than contribute to the transmission of forces (14,15). However, precise roles of decorin are not well understood, partly because its three-dimensional structure is unresolved.The structure of decorin GAG chains among collagen fibrils was observed by ultrathin sections of a sample stained with Cupromeronic blue and the critical electrolyte concentration technique, which was used to reveal the sulfated GAGs under a transmission electron microscope (TEM) (5, 16). Scott et al. (17) proposed the "shape module model" in which the decorin ...

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“…Observation of the surface of resin embedded biological samples using backscattered electron imaging of SEM provides an image that is similar to TEM results (19,20 …”

supporting

confidence: 60%

supporting

confidence: 57%

Ring-Mesh Model of Proteoglycan Glycosaminoglycan Chains in Tendon based on Three-dimensional Reconstruction by Focused Ion Beam Scanning Electron Microscopy

Watanabe

Kametani

Koyama

et al. 2016

Journal of Biological Chemistry

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“…However, the geometrical nature of these structures must be confirmed through serial sections rather than observed on a single section. In fact, FIB-SEM has been recently employed to assess the three-dimensional aspects of several tissues using a specific imaging software (3,(25)(26)(27), and also in dental histology, where it revealed the connections among dentinal tubules (28). One limitation of this study is that only two-dimensional images were used; that being said, we are currently refining one three-dimensional FIB-SEM reconstruction protocol that will be presented to the bone research community in a near future.…”

Section: Discussionmentioning

confidence: 99%

Biological application of focus ion beam-scanning electron microscopy (FIB-SEM) to the imaging of cartilaginous fibrils and osteoblastic cytoplasmic processes

Hasegawa

Endo

Tsuchiya

et al. 2017

Journal of Oral Biosciences

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Objectives: The aim of this study is a biological application of focused ion beam-scanning electron microscopy (FIB-SEM) to demonstrate serial sectional images of skeletal tissues, here presenting the ultrastructure of 1) cartilaginous extracellular fibrils and 2) osteoblastic cytoplasmic processes.Methods: Seven weeks-old female wild-type mice were fixed with half-Karnovsky solution and subsequent OsO4, and the tibiae were extracted for block staining prior to observation under transmission electron microscope (TEM) and FIB-SEM.Results: TEM showed the fine fibrillar, but somewhat amorphous ultrastructure of the intercolumnar septa in the growth plate cartilage. Alternatively, FIB-SEM revealed bundles of stout fibrils at regular intervals paralleling the septa's longitudinal axis, as well as vesicular structures embedded in the cartilaginous matrix of the proliferative zone. In the primary trabeculae, both TEM and FIB-SEM showed several osteoblastic cytoplasmic processes on the osteoid, with numbers higher than those seen in the bone matrix. FIB-SEM revealed the agglomeration of cytoplasmic processes beneath the osteoblasts, which formed a tubular continuum extending from those cells. Based on these findings, we postulated that osteoblasts not only extend their cytoplasmic processes through to the bone matrix, but also stack these cell processes on the osteoid of the primary trabeculae. Conclusion:Taken together, it is likely that FIB-SEM imaging strategy on serial sections may successfully deliver new insights on the ultrastructure of cartilage and bone tissues. words

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“…Recently, serial slice scanning electron microscopy are getting popular for 3D reconstruction of animal tissues 23), 24) . Although these new approaches are very useful, the resolution of the images is inferior to transmission electron microscopy of ultrathin sections.…”

Section: Other Means For Structome Analysis Than Serial Ultrathin Secmentioning

confidence: 99%

An electron microscopic study of microorganisms: from influenza virus to deep-sea microorganisms

Yamaguchi

2015

Proc. Jpn. Assoc. Mycotoxicol.

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Three topics from electron microscopic studies of microorganisms carried out in my laboratory in recent ten years are described. 1) Influenza A virus was observed in water by an ice-embedding method using phase contrast electron microscopy developed in Japan. Virions appeared as spherical or elongated particles consisting of spikes, an envelope, and a core with high contrast. 2) A new term the "structome" was introduced and defined as "the quantitative and three-dimensional structural information of a whole cell at electron microscopic level." We performed structome analyses of Saccharomyces cerevisiae by using freeze-substitution and serial ultrathin sectioning electron microscopy. We found that there were one to four mitochondria and about 195,000 ribosomes in a cell. 3) In the deep-sea off the coast of Japan, we discovered a unique microorganism appearing to have cellular features intermediate between prokaryotes and eukaryotes. The organism, named as the Myojin parakaryote, was two orders of magnitude larger than a typical bacterium and had a large "nucleoid", surrounded by a single layered "nucleoid membrane", and bacteria-like "endosymbionts", but it lacked mitochondria. This organism exemplifies a potential evolutionary path between prokaryotes and eukaryotes, and the presence of the organism supports the endosymbiotic theory for the origin of mitochondria and the karyogenetic hypothesis for the origin of the nucleus. These studies show that the electron microscopy is a powerful tool for studying a wide range of problems of microorganisms.

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Beam deceleration for block-face scanning electron microscopy of embedded biological tissue

Cited by 98 publications

References 26 publications

Ring-Mesh Model of Proteoglycan Glycosaminoglycan Chains in Tendon based on Three-dimensional Reconstruction by Focused Ion Beam Scanning Electron Microscopy

Ring-Mesh Model of Proteoglycan Glycosaminoglycan Chains in Tendon based on Three-dimensional Reconstruction by Focused Ion Beam Scanning Electron Microscopy

Biological application of focus ion beam-scanning electron microscopy (FIB-SEM) to the imaging of cartilaginous fibrils and osteoblastic cytoplasmic processes

An electron microscopic study of microorganisms: from influenza virus to deep-sea microorganisms

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