Volume Scanning Electron Microscopy for 3D Analysis of Biological Ultrastructure

Miyaki, Takayoshi; Kawasaki, Yuto; Hosoyamada, Yasue; Amari, Takashi; Kinoshita, Mui; Matsuda, Hironori; Kakuta, Soichiro; Sakai, Tatsuo; Ichimura, Koichiro

doi:10.14789/jmj.2020.66.jmj19-r19

Cited by 4 publications

(4 citation statements)

References 37 publications

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“…Focused-ion beam-SEM tomography was performed as described previously ( Miyaki et al, 2020a , b ; Figure 2 ). In brief, the cortex of the fixed kidney was cut into 250-μm-thick slices with a DTK-1000 MicroSlicers (Dosaka EM, Kyoto, Japan).…”

Section: Methodsmentioning

confidence: 99%

“…These FIB-SEM images are comparable to conventional transmission electron microscopy (TEM) images. Moreover, three-dimensional (3D) cellular structures can be reconstructed from serial sectional images with high reliability ( Miyaki et al, 2020a , b ). Recently, using this approach, our group has re-evaluated the 3D ultrastructure of podocytes in the context of health ( Ichimura et al, 2015 ), development ( Ichimura et al, 2017 ), disease ( Ichimura et al, 2019 ), and evolution ( Ichimura and Sakai, 2017 ; Kawasaki et al, 2019 ; Miyaki et al, 2020c ).…”

Section: Introductionmentioning

confidence: 99%

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Three-Dimensional Architecture of Glomerular Endothelial Cells Revealed by FIB-SEM Tomography

Kawasaki

Hosoyamada

Miyaki

et al. 2021

Front. Cell Dev. Biol.

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Focused-ion beam-scanning electron microscopic (FIB-SEM) tomography enables easier acquisition of a series of ultrastructural, sectional images directly from resin-embedded biological samples. In this study, to clarify the three-dimensional (3D) architecture of glomerular endothelial cells (GEnCs) in adult rats, we manually extracted GEnCs from serial FIB-SEM images and reconstructed them on an Amira reconstruction software. The luminal and basal surface structures were clearly visualized in the reconstructed GEnCs, although only the luminal surface structures could be observed by conventional SEM. The luminal surface visualized via the reconstructed GEnCs was quite similar to that observed through conventional SEM, indicating that 3D reconstruction could be performed with high accuracy. Thus, we successfully described the 3D architecture of normal GEnCs in adult rats more clearly and precisely than ever before. The GEnCs were found to consist of three major subcellular compartments, namely, the cell body, cytoplasmic ridges, and sieve plates, in addition to two associated subcellular compartments, namely, the globular protrusions and reticular porous structures. Furthermore, most individual GEnCs made up a “seamless” tubular shape, and some of them formed an autocellular junction to make up a tubular shape. FIB-SEM tomography with reconstruction is a powerful approach to better understand the 3D architecture of GEnCs. Moreover, the morphological information revealed in this study will be valuable for the 3D pathologic evaluation of GEnCs in animal and human glomerular diseases and the structural analysis of developmental processes in the glomerular capillary system.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Three-Dimensional Architecture of Glomerular Endothelial Cells Revealed by FIB-SEM Tomography

Kawasaki

Hosoyamada

Miyaki

et al. 2021

Front. Cell Dev. Biol.

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“…Subsequently, samples were dehydrated with a graded series of ethanol and embedded in epoxy resin, Oken Epok 812 (Okenshoji, Tokyo, Japan). For the detailed protocol of sample preparation, see Miyaki et al ( 2020a , b ).…”

Section: Methodsmentioning

confidence: 99%

Nephrocytes are part of the spectrum of filtration epithelial diversity

et al. 2020

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The excretory system produces urine by ultrafiltration via a filtration epithelium. Podocytes are widely found as filtration epithelial cells in eucoelomates. In some animal taxa, including insects and crustaceans, nephrocytes serve to separate toxic substances from the body fluid, in addition to podocytes. Drosophila nephrocytes have been recently utilized as a model system to study podocyte function and disease. However, functionality and cellular architecture are strikingly different between Drosophila nephrocytes and eucoelomate podocytes, and the phylogenetic relationship between these cells remains enigmatic. In this study, using focused-ion beam-scanning electron microscopy (FIB-SEM) tomography, we revealed three-dimensional architecture of decapod nephrocytes with unprecedented accuracy—they filled an enormous gap, which can be called “missing link,” in the evolutionary diversity of podocytes and nephrocytes. Thus, we concluded that nephrocytes are part of the spectrum of filtration epithelial diversity in animal phylogeny.

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“…The coloring of individual podocytes in the FE-SEM images is useful for understanding the architecture and mutual relationship of the podo-cytes ( Figure-2Bʼ). Such coloring can be easily performed by commercially available image software, such as Adobe Photoshop or Illustrator 10) .…”

Section: The Procedures Of Fe-sem Analysis Of Podocytesmentioning

confidence: 99%

Application of Conventional FE-SEM to Podocyte Structural Analysis: Protocol and Usage Examples

Kawasaki

Miyaki

Yamaguchi

et al. 2021

Juntendo Medical Journal

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Podocytes, an epithelial cell type specialized for glomerular filtration, exhibit a complex projection structure. Field-emission scanning electron microscopy (FE-SEM) is a powerful approach to observing the surface structure of podocytes, particularly their pathologic alterations, including foot process effacement. Here, we introduce the conventional FE-SEM protocol for three-dimensional visualization of podocyte surface structure in our institution. This method is reproducible and allows us to visualize the luminal surface of numerous podocytes simultaneously.

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Volume Scanning Electron Microscopy for 3D Analysis of Biological Ultrastructure

Abstract: TAKAYUKI MIYAKI ＊1) , YUTO KAWASAKI ＊1) , YASUE HOSOYAMADA ＊1) 2) , TAKASHI AMARI ＊1) , MUI KINOSHITA ＊1) , HIRONORI MATSUDA ＊1) , SOICHIRO KAKUTA ＊3) , TATSUO SAKAI ＊1) , KOICHIRO ICHIMURA ＊1) 3)

Cited by 4 publications

References 37 publications

Three-Dimensional Architecture of Glomerular Endothelial Cells Revealed by FIB-SEM Tomography

Three-Dimensional Architecture of Glomerular Endothelial Cells Revealed by FIB-SEM Tomography

Nephrocytes are part of the spectrum of filtration epithelial diversity

Application of Conventional FE-SEM to Podocyte Structural Analysis: Protocol and Usage Examples

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