Field-Emission Scanning Electron Microscope as a Tool for Large-Area and Large-Volume Ultrastructural Studies

SIMPLE SUMMARY: Ultrastructural studies of cells and tissues are usually performed using transmission electron microscopy (TEM), which enables imaging at the highest possible resolution. The weak point of TEM is the limited ability to analyze the ultrastructure of large areas and volumes of biological samples. This limitation can be overcome by using modern field-emission scanning electron microscopy (FE-SEM) with high-sensitivity detection, which enables the creation of TEM-like images from the flat surfaces of resin-embedded biological specimens. Several FE-SEM-based techniques for two- and three-dimensional ultrastructural studies of cells, tissues, organs, and organisms have been developed in the 21st century. These techniques have created a new era in structural biology and have changed the role of the scanning electron microscope (SEM) in biological and medical laboratories. Since the premiere of the first commercially available SEM in 1965, these instruments were used almost exclusively to obtain topographical information over a large range of magnifications. Currently, FE-SEM offers many attractive possibilities in the studies of cell and tissue ultrastructure, and they are presented in this review. ABSTRACT: The development of field-emission scanning electron microscopes for high-resolution imaging at very low acceleration voltages and equipped with highly sensitive detectors of backscattered electrons (BSE) has enabled transmission electron microscopy (TEM)-like imaging of the cut surfaces of tissue blocks, which are impermeable to the electron beam, or tissue sections mounted on the solid substrates. This has resulted in the development of methods that simplify and accelerate ultrastructural studies of large areas and volumes of biological samples. This article provides an overview of these methods, including their advantages and disadvantages. The imaging of large sample areas can be performed using two methods based on the detection of transmitted electrons or BSE. Effective imaging using BSE requires special fixation and en bloc contrasting of samples. BSE imaging has resulted in the development of volume imaging techniques, including array tomography (AT) and serial block-face imaging (SBF-SEM). In AT, serial ultrathin sections are collected manually on a solid substrate such as a glass and silicon wafer or automatically on a tape using a special ultramicrotome. The imaging of serial sections is used to obtain three-dimensional (3D) information. SBF-SEM is based on removing the top layer of a resin-embedded sample using an ultramicrotome inside the SEM specimen chamber and then imaging the exposed surface with a BSE detector. The steps of cutting and imaging the resin block are repeated hundreds or thousands of times to obtain a z-stack for 3D analyses.