Sub-Surface Serial Block Face Scanning Electron Microscopy

Abstract

Serial block face scanning electron microscopy (SBF-SEM) provides nanoscale 3D ultrastructure of tissue 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 used Monte Carlo simulations of SEM images from an epoxy block containing 5-nm diameter carbon spheres stained with 14% osmium positioned at different depths, as a model for small biological structures. A linear relationship was found between the depth of the spheres and the ratio of backscattered signals at primary beam energies of 1.4 keV and 6.8 keV, which allowed us to generate 3D tomograms with a depth resolution of around 5 nm. Experiments are in progress to test this technique using a Zeiss Sigma-VP SEM equipped with a Gatan 3View SBF system. Sub-surface SBF-SEM could potentially match focused ion beam (FIB) SEM in terms of z-resolution, but with the added advantage of providing higher throughput and larger tissue volumes. The research was supported by the intramural program of NIBIB.