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

Koichiro Ichimura,Keisuke Ohta,Naoyuki Miyazaki,Masato Koike,Tatsuo Sakai,Kei-Ichiro Nakamura,Kazuyoshi Murata,Shoji Sadayama

doi:10.1038/srep08993

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.

Highlights

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
It was previously believed that podocytes are divided into three kinds of subcellular compartment: cell body, primary process, and foot process, which are 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

Summary

Introduction

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. A block-face image can be obtained directly from a smooth surface of resin-embedded specimen using SEM, and the contrast-inverted block-face image is quite similar to the conventional TEM image of an ultrathin section[11,12,13] Such SEM images can be serially and automatically acquired in a depth direction using a special SEM equipped with a system to remove a thin layer from the resin-embedded sample. Threedimensional reconstruction of subcellular structures based on serial block-face images has been widely applied in a variety of biomedical research fields, especially neuroscience This method should be useful for the structural analysis of renal tissues and cells including podocytes, but there has been no application of this method in this field, except for a recent study which clearly confirmed the glycocalyx layer of glomerular endothelial cells and the subpodocyte space[14]

Methods

Results

Conclusion