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Three-dimensional architecture of podocytes revealed by block-face scanning electron microscopy.

ABSTRACT: Block-face imaging is a scanning electron microscopic technique which enables easier acquisition of serial ultrastructural images directly from the surface of resin-embedded biological samples with a similar quality to transmission electron micrographs. In the present study, we analyzed the three-dimensional architecture of podocytes using serial block-face imaging. It was previously believed that podocytes are divided into three kinds of subcellular compartment: cell body, primary process, and foot process, which are simply aligned in this order. When the reconstructed podocytes were viewed from their basal side, the foot processes were branched from a ridge-like prominence, which was formed on the basal surface of the primary process and was similar to the usual foot processes in structure. Moreover, from the cell body, the foot processes were also emerged via the ridge-like prominence, as found in the primary process. The ridge-like prominence anchored the cell body and primary process to the glomerular basement membrane, and connected the foot processes to the cell body and primary process. In conclusion, serial block-face imaging is a powerful tool for clear understanding the three-dimensional architecture of podocytes through its ability to reveal novel structures which were difficult to determine by conventional transmission and scanning electron microscopes alone.

SUBMITTER: Ichimura K

PROVIDER: S-EPMC4355681 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Three-dimensional architecture of podocytes revealed by block-face scanning electron microscopy.

Ichimura Koichiro K Miyazaki Naoyuki N Sadayama Shoji S Murata Kazuyoshi K Koike Masato M Nakamura Kei-Ichiro K Ohta Keisuke K Sakai Tatsuo T

Scientific reports 20150311

Block-face imaging is a scanning electron microscopic technique which enables easier acquisition of serial ultrastructural images directly from the surface of resin-embedded biological samples with a similar quality to transmission electron micrographs. In the present study, we analyzed the three-dimensional architecture of podocytes using serial block-face imaging. It was previously believed that podocytes are divided into three kinds of subcellular compartment: cell body, primary process, and ...[more]

PMID: 25759085

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Project description:Serial block face scanning electron microscopy (SBF-SEM) is a relatively new technique that allows the acquisition of serially sectioned, imaged and digitally aligned ultrastructural data. There is a wealth of information that can be obtained from the resulting image stacks but this presents a new challenge for researchers - how to computationally analyse and make best use of the large datasets produced. One approach is to reconstruct structures and features of interest in 3D. However, the software programmes can appear overwhelming, time-consuming and not intuitive for those new to image analysis. There are a limited number of published articles that provide sufficient detail on how to do this type of reconstruction. Therefore, the aim of this paper is to provide a detailed step-by-step protocol, accompanied by tutorial videos, for several types of analysis programmes that can be used on raw SBF-SEM data, although there are more options available than can be covered here. To showcase the programmes, datasets of skeletal muscle from foetal and adult guinea pigs are initially used with procedures subsequently applied to guinea pig cardiac tissue and locust brain. The tissue is processed using the heavy metal protocol developed specifically for SBF-SEM. Trimmed resin blocks are placed into a Zeiss Sigma SEM incorporating the Gatan 3View and the resulting image stacks are analysed in three different programmes, Fiji, Amira and MIB, using a range of tools available for segmentation. The results from the image analysis comparison show that the analysis tools are often more suited to a particular type of structure. For example, larger structures, such as nuclei and cells, can be segmented using interpolation, which speeds up analysis; single contrast structures, such as the nucleolus, can be segmented using the contrast-based thresholding tools. Knowing the nature of the tissue and its specific structures (complexity, contrast, if there are distinct membranes, size) will help to determine the best method for reconstruction and thus maximize informative output from valuable tissue.

Dataset Information

Three-dimensional architecture of podocytes revealed by block-face scanning electron microscopy.

Publications

Three-dimensional architecture of podocytes revealed by block-face scanning electron microscopy.

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