Developments in scanning electron microscopes (SEM) in recent years have led to ground-breaking opportunities in volume imaging of cells and tissues. The aim of this EMBO Practical Course is to provide extensive theoretical and practical knowledge on the three main techniques for automated serial imaging based on SEM (ASI-SEM): microtome-based serial block-face SEM, focused ion beam SEM and array tomography. At the heart of the course, participants will be able to assess the potential of each technique to understand which fits best with their own scientific project. The participants will acquire the theoretical background to understand the different techniques as well as how to optimize the imaging conditions. Data processing, analysis and management, key considerations in all ASI-SEM techniques will also be covered. This EMBO Practical Course has a significant hands-on component where students participate in experimentation on i) sample preparation adapted to each ASI-SEM technique ii) operation of the hardware (SEMs, automated microtomes) and iii) image processing software (stack registration, segmentation, tracing).