Abstract
Serial block-face electron microscopy (SBEM) provides nanoscale 3D ultrastructure of embedded and stained cells and tissues in volumes of up to 107 µm3. 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.
Highlights
Over the past decade, techniques have been developed, which enable nanoscale 3D imaging of large biological tissue volumes[1,2,3,4,5,6,7,8,9,10]
Z=0 A(z, Em)S(x, y, z)dz where B(x, y, Em) is the number of electrons per unit area backscattered from the specimen at position x, y for landing energy Em; S(x, y, z) is the 3D distribution of heavy-atom stain density in the specimen block; and A(z, Em) describes the interaction of the incident electrons of landing energy Em with the plastic embedding material containing heavy atoms located at depth z
Equation 1 only includes electrons that are backscattered from the heavy atoms of the stain and excludes electrons that are backscattered from the uniform plastic embedding material, since those contribute a constant background to the backscattering coefficient, which can be readily subtracted
Summary
Techniques have been developed, which enable nanoscale 3D imaging of large biological tissue volumes[1,2,3,4,5,6,7,8,9,10]. We explore the feasibility of improving the z-resolution in SBEM by acquiring backscattered electron images with different primary energies (Fig. 1c,e) so that the incident electron probe is concentrated at different subsurface depths, which provides subslice resolution (Fig. 1f) This idea has been described by Boughorbel et al.[22] and by de Goede et al.[23], who developed a technique (ThruSight) for FEI/Thermo Fisher Scientific SEMs24, based on a blind deconvolution method that iteratively fits a series of images recorded at different primary beam energies to deduce the most likely 3D subsurface structure. Our present aim is to obtain a complementary physically based model incorporating electron scattering theory through a Monte Carlo simulation, which enables us to reconstruct subsurface features, to estimate errors in the reconstruction, and to determine the fundamental limits of subsurface imaging that are imposed by radiation damage, which results in collapse of the specimen under electron irradiation
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