Sample Processing and Benchmarking for Multibeam Optical Scanning Transmission Electron Microscopy

Abstract 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.