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

Scientific Reports
Q HeR D Leapman

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.

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Citations

Jun 20, 2020·Stem Cell Reviews and Reports·Rahele A FarahaniAlfonso Eirin
Jan 30, 2020·BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology·Thomas CremerChristoph Cremer
Sep 18, 2020·The Journal of Biological Chemistry·Peter O BayguinovJames A J Fitzpatrick
Dec 23, 2020·Microscopy and Microanalysis : the Official Journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada·Yoram VosJacob P Hoogenboom

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Methods Mentioned

BETA
electron
scanning electron microscopy
transmission electron microscopy
electron scattering
imaging technique
electron microscopy

Software Mentioned

INCA microanalysis
IMOD
Amira
DTSA
Fortran
Teneo VolumeScope
ThruSight
CASINO
ImageJ
SBEM

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