Reconstructing adhesion structures in tissues by cryo-electron tomography of vitrified frozen sections

Abstract

Cryo-electron tomography enables three-dimensional insights into the macromolecular architecture of cells in a close-to-life state. However, it is limited to thin specimens, <1.0 μm in thickness, typically restricted to the peripheral areas of intact eukaryotic cells. Analysis of tissue ultrastructure, on the other hand, requires physical sectioning approaches, preferably cryo-sectioning, following which electron tomography (ET) may be performed. Nevertheless, cryo-electron microscopy of vitrified sections is a demanding technique and typically cannot be used to examine thick sections, >80-100 nm, due to surface crevasses. Here, we explore the potential use of cryo-ET of vitrified frozen sections (VFSs) for imaging cell adhesions in chicken smooth muscle and mouse epithelial tissues. By investigating 300-400 nm thick sections, which are collected on the EM grid and re-vitrified, we resolved fine 3D structural details of the membrane-associated dense plaques and flanking caveoli in smooth muscle tissue, and desmosomal adhesions in stratified epithelium. Technically, this method offers a simple approach for reconstructing thick volumes of hydrated frozen sections.