Electron microscopy (EM) is an indispensable technique to visualize biological ultrastructure in health and disease. High-throughput EM further enables larger scales and volumes to be recorded within feasible timeframes. Multibeam optical scanning transmission EM (OSTEM) utilizes multiple beamlets and optical separation of the transmitted electrons to increase imaging throughput with transmission-based imaging. However, the compatibility of multibeam OSTEM with routine sample preparation protocols and the effect of machine settings on image quality remain largely unknown. Here, we show multibeam OSTEM to be an order of magnitude faster than (scanning) transmission EM while yielding comparable high-quality images of tissue processed with standard high-contrast staining protocols. Multibeam OSTEM benefits from embedding approaches that introduce high contrast but is flexible in the type of stain used. Optimal results are obtained using an acceleration voltage of 5 kV, where section thickness and pixel dwell time require a balance between throughput and image quality. Our results show high-throughput EM with imaging quality comparable with commonly used transmission-based modalities, enabling biological ultrastructure analysis across larger scales and volumes.@en

Elucidating the 3D nanoscale structure of tissues and cells is essential for understanding the complexity of biological processes. Electron microscopy (EM) offers the resolution needed for reliable interpretation, but the limited throughput of electron microscopes has hindered its ability to effectively image large volumes. We report a workflow for volume EM with FAST-EM, a novel multibeam scanning transmission electron microscope that speeds up acquisition by scanning the sample in parallel with 64 electron beams. FAST-EM makes use of optical detection to separate the signals of the individual beams. The acquisition and 3D reconstruction of ultrastructural data from multiple biological samples is demonstrated. The results show that the workflow is capable of producing large reconstructed volumes with high resolution and contrast to address biological research questions within feasible acquisition time frames.@en