Serial block face scanning electron microscopy in cell biology: Applications and technology

•SBFSEM was introduced in 2004 and has become a major tool for three-dimensional electron microscopy.•SBFSEM has become widespread in connectomics, cellular and matrix biology.•Problems with the technique are being overcome by advances in technology.•Data analysis represents a bottleneck but advances in computation and crowd-sourcing are set to improve this. Three-dimensional electron microscopy (3DEM) is an imaging field containing several powerful modalities such as serial section transmission electron microscopy and electron tomography. However, large-scale 3D studies of biological ultrastructure on a cellular scale have historically been hampered by the difficulty of available techniques. Serial block face scanning electron microscopy (SBFSEM) is a 3DEM technique, developed in 2004, which has greatly increased the reliability, availability and throughput of 3DEM. SBFSEM allows for 3D imaging at resolutions high enough to resolve membranes and small vesicles whilst having the capability to collect data with a large field of view. Since its introduction it has become a major tool for ultrastructural investigation and has been applied in the study of many biological fields, such as connectomics, cellular and matrix biology. In this review, we will discuss biological SBFSEM from a technical standpoint, with a focus on cellular applications and also subsequent image analysis techniques.