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.

A flexible, multilayered protein scaffold maintains the slit in between glomerular podocytes

“…Recently, two other groups reported results using block-face SEM (FIB-SEM) where they could resolve the arrangement of neighboring podocyte foot processes and prove that individual podocytes indeed only interdigitate with adjacent podocytes but not with their own processes (33, 34). However, this technique does not have the resolution to study the SD in detail.…”

Clarifying Tissue Clearing

“…Another possibility is to image the surface of a block of tissue and then sequentially shave off the surface. Such “blockface” methods are used in both in light (Toga et al, 1994; Tsai et al, 2009a) and electron microscopy (Denk and Horstmann, 2004; Ichimura et al, 2015). Blockface approaches eliminate the loss and alignment issues of sections but are destructive in the sense that once each section is imaged it is destroyed to reveal the next block surface.…”

New approaches in renal microscopy

“…This method was used by the Allen Brain Institute to image both human [6,7] and mouse brain [8]. In the kidney, scanning EM with serial sectioning [9] was used to image the whole human glomerulus in three-dimensions [10] along with the basal surface of podocytes within rat glomeruli [11]. …”

“…It provides superb resolution, but is technically challenging and more expensive than other EM techniques. Recently, focused ion beam-scanning electron microscopy (FIB-SEM) has been used to image podocytes and the glycocalyx with excellent resolution [10,11,53]. FIB-SEM is capable of providing global high-resolution three-dimensional EM images of the area of interest.…”