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

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.