Titze B Genoud C 2016 Electron microscopy (EM) has been a key imaging method to investigate biological ultrastructure for over six decades. In recent years, novel volume EM techniques have significantly advanced nanometre-scale imaging of cells and tissues in three dimensions. Previously, this had depended on the slow and error-prone manual tasks of cutting and handling large numbers of sections, and imaging them one-by-one with transmission EM. Now, automated volume imaging methods mostly based on scanning EM (SEM) allow faster and more reliable acquisition of serial images through tissue volumes and achieve higher z-resolution. Various software tools have been developed to manipulate the acquired image stacks and facilitate quantitative analysis. Here, we introduce three volume SEM methods: serial block-face electron microscopy (SBEM), focused ion beam SEM (FIB-SEM) and automated tape-collecting ultramicrotome SEM (ATUM-SEM). We discuss and compare their capabilities, provide an overview of the full volume SEM workflow for obtaining 3D datasets and showcase different applications for biological research. https://sonar.ch/global/documents/201671 eng info:eu-repo/semantics/altIdentifier/doi/10.1111/boc.201600024 info:eu-repo/semantics/altIdentifier/pmid/27432264 Biology of the cell. - 2016 Brain/nervous system Cellular imaging Electron microscopy Systems biology Animals Brain Histocytological Preparation Techniques Humans Image Processing, Computer-Assisted Microscopy, Electron, Scanning Microtomy Volume scanning electron microscopy for imaging biological ultrastructure. http://purl.org/coar/resource_type/c_6501