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Serial block-face electron microscopy (SBEM) provides nanoscale 3D ultrastructure of embedded and stained cells and tissues in volumes of up to 10 7  µm 3 . In SBEM, electrons with 1–3 keV energies are incident on a specimen block, from which backscattered electron (BSE) images are collected with  x ,  y  resolution of 5–10 nm in the block-face plane, and successive layers are removed by an  in situ  ultramicrotome. Spatial resolution along the  z- direction, however, is limited to around 25 nm by the minimum cutting thickness. To improve the  z- resolution, we have extracted depth information from BSE images acquired at dual primary beam energies, using Monte Carlo simulations of electron scattering. The relationship between depth of stain and ratio of dual-energy BSE intensities enables us to determine 3D structure with a ×2 improvement in  z- resolution. We demonstrate the technique by sub-slice imaging of hepatocyte membranes in liver tissue.

Biological serial block face scanning electron microscopy at improved z-resolution based on Monte Carlo model