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

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          Abstract

          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.

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          CASINO V2.42: a fast and easy-to-use modeling tool for scanning electron microscopy and microanalysis users.

          Monte Carlo simulations have been widely used by microscopists for the last few decades. In the beginning it was a tedious and slow process, requiring a high level of computer skills from users and long computational times. Recent progress in the microelectronics industry now provides researchers with affordable desktop computers with clock rates greater than 3 GHz. With this type of computing power routinely available, Monte Carlo simulation is no longer an exclusive or long (overnight) process. The aim of this paper is to present a new user-friendly simulation program based on the earlier CASINO Monte Carlo program. The intent of this software is to assist scanning electron microscope users in interpretation of imaging and microanalysis and also with more advanced procedures including electron-beam lithography. This version uses a new architecture that provides results twice as quickly. This program is freely available to the scientific community and can be downloaded from the website: (www.gel.usherb.ca/casino).
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            elsepa—Dirac partial-wave calculation of elastic scattering of electrons and positrons by atoms, positive ions and molecules

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              Volume scanning electron microscopy for imaging biological ultrastructure.

              Electron microscopy (EM) has been a key imaging method to investigate biological ultrastructure for over six decades. In recent years, novel volume EM techniques have significantly advanced nanometre-scale imaging of cells and tissues in three dimensions. Previously, this had depended on the slow and error-prone manual tasks of cutting and handling large numbers of sections, and imaging them one-by-one with transmission EM. Now, automated volume imaging methods mostly based on scanning EM (SEM) allow faster and more reliable acquisition of serial images through tissue volumes and achieve higher z-resolution. Various software tools have been developed to manipulate the acquired image stacks and facilitate quantitative analysis. Here, we introduce three volume SEM methods: serial block-face electron microscopy (SBEM), focused ion beam SEM (FIB-SEM) and automated tape-collecting ultramicrotome SEM (ATUM-SEM). We discuss and compare their capabilities, provide an overview of the full volume SEM workflow for obtaining 3D datasets and showcase different applications for biological research.
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                Author and article information

                Contributors
                leapmanr@mail.nih.gov
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                28 August 2018
                28 August 2018
                2018
                : 8
                : 12985
                Affiliations
                [1 ]ISNI 0000 0004 0533 5934, GRID grid.280347.a, National Institute of Biomedical Imaging and Bioengineering, NIH, ; Bethesda, MD 20892 USA
                [2 ]Gatan, Inc., 5794W. Las Positas Blvd, Pleasanton, CA 94588 USA
                [3 ]ISNI 0000 0001 2315 1184, GRID grid.411461.7, Department of Material Science and Engineering, , University of Tennessee, ; Knoxville, TN 37996 USA
                [4 ]ISNI 0000 0004 0446 2659, GRID grid.135519.a, Center for Nanophase Material Sciences, , Oak Ridge National Laboratory, ; Oak Ridge, TN 37831 USA
                Article
                31231
                10.1038/s41598-018-31231-w
                6113311
                30154532
                8ed01d79-f540-47d8-bbcf-6dee36fa1e55
                © The Author(s) 2018

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 10 January 2018
                : 10 August 2018
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