Preparing large biological specimens for high-throughput volume electron microscopy

Abstract To enable high-throughput volume electron microscopy (vEM) imaging, osmium-based staining methods are routinely employed to enhance membrane contrast and electrical conductivity in biological specimens. Nevertheless, reliable preparation of densely and uniformly stained tissue blocks remains a major challenge for large-scale vEM-based ultrastructure analysis, especially for connectomic studies. Recently, multiple strategies have been proposed to achieve high-quality en bloc staining of millimeter-sized brain samples without compromising membrane contrast, paving the way for system-level neural circuit mapping using vEM. Herein, we review the development of large-volume tissue staining and elaborate the underlying chemical logic based on cumulative experimental evidence. We further discuss emerging challenges associated with adapting current staining strategies for cryo-preserved tissue samples and in-resin applications of correlative light and electron microscopy.