Sub-slice resolution in serial block face SEM of biological structures at near isotropic spatial resolution using multiple beam energies and Monte Carlo simulations

Authors

• Q He
• G Zhang
• RD Leapman

Abstract

Serial block face (SBF) imaging in the scanning electron microscope (SEM) provides nanoscale 3D ultrastructure of biological samples up to several hundred micrometers in size. In SBF-SEM, an ultramicrotome built into the SEM specimen stage successively removes thin sections from a plastic-embedded, heavy metal-stained specimen. After each cut, the freshly exposed block face is imaged at a low incident electron energy using the backscattered electron signal, which is sensitive to heavy atoms in the sample. Although the x-y resolution in the plane of the block face is approximately 5 nm, the resolution along the z-axis in SBF-SEM is limited by the minimum slice thickness of around 25 nm. We have explored the feasibility of improving the z-resolution in SBF-SEM by recording images at more than one primary beam energy, thus sampling different depths below the block surface. We performed Monte Carlo simulations of SEM backscattered electron images from an epoxy block containing stained subvolumes as a model for small biological structures within cells. The model obtained from the simulation was tested experimentally on embedded blocks of brain tissue using an SEM equipped with a field emission electron source and an SBF system to demonstrate that 3D ultrastructure could be determined from layers that are half the thickness of the slices cut by the microtome knife. Thus, the z-resolution in SBF-SEM becomes competitive with focused ion beam (FIB)-SEM, but can be performed at a faster rate from larger volumes.

Scientific Focus Area: Biomedical Engineering and Biophysics

This page was last updated on Friday, March 26, 2021