Three-dimensional imaging of podocyte ultrastructure using FE-SEM and FIB-SEM tomography

Abstract

Podocytes are specialized epithelial cells used for glomerular filtration in the kidney. They can be divided into the cell body, primary process and foot process. Here, we describe two useful methods for the three-dimensional(3D) visualization of these subcellular compartments in rodent podocytes. The first method, field-emission scanning electron microscopy (FE-SEM) with conductive staining, is used to visualize the luminal surface of numerous podocytes simultaneously. The second method, focusedion beam SEM (FIB-SEM) tomography, allows the user to obtain serial images from different depths of field, or Z-stacks, of the glomerulus. This allows for the 3D reconstruction of podocyte ultrastructure, which can be viewed from all angles, from a single image set. This is not possible with conventional FE-SEM. The different advantages and disadvantages of FE-SEM and FIB-SEM tomography compensate for the weaknesses of the other. The combination renders a powerful approach for the 3D analysis of podocyte ultrastructure. As a result, we were able to identify a new subcellular compartment of podocytes, "ridge-like prominences" (RLPs).

3D Visualization of the Podocyte Actin Network using Integrated Membrane Extraction, Electron Microscopy, and Deep Learning

“…Because SEM only allows visualization of surface topology, we sought a method to view the whole depth of the GFB. To achieve this, we used FIB-SEM, which generates an image stack in the Zdirection, thus providing a view of the structures located deep in the tissue [27]. Results revealed that the central actin cables in the foot processes were connected with the neighboring slit diaphragms located just above the GBM, forming a continuous structure (Fig.…”

“…Transmission electron microscopy (TEM) and field emission-scanning electron microscopy (SEM) have revealed broadening of podocyte foot processes as they efface [19][20][21] and changes to endothelial cells associated with injury in specific glomerular diseases [22]. Two recently-developed techniques, block-face (BF)-SEM [23,24] and focused ion beam (FIB)-SEM [25][26][27][28], have uncovered key aspects of glomerular and podocyte ultrastructure in 3D, including ridge-like promenades [25], the irregular GBM of Alport syndrome [23], podocyteparietal cell interconnectivity [29], and changes to podocytes during the FPE that follows injury [27].…”

Volume Scanning Electron Microscopy for 3D Analysis of Biological Ultrastructure

“…The stained samples were dehydrated and embedded in epoxy-resin by using standard protocol. For the detailed protocol of sample processing for volume SEM, see Miyaki et al 17) .…”

“…As a result of these features, a far higher depth resolution of a reconstruction image can be obtained from the serial images developed by FIB-SEM than by DK-SEM. 17) . The authors have permission to copy from the copyright owner.…”

“…E: endothelial cell; M: mesangial cell; and P: podocyte. (This figure was partially reprinted from Miyaki T et al: Cell Tissue Res, 2020; 379: 245-25417) . The authors have permission to copy from the copyright owner.…”