doi:10.1017/S1551929516000730 28 www.microscopy-today.com • 2016 September A Reverse Engineering Approach for Imaging Neuronal Architecture – Large-Area, High-Resolution SEM Imaging Christine A. Brantner, 1 * Martin Rasche, 4 Kevin E. Burcham, 3 Joseph Klingfus, 3 Joel Fridmann, 3 Jason E. Sanabia, 3 Can E. Korman, 2 and Anastas Popratiloff 1 1 GW Nanofabrication and Imaging Center, Te George Washington University, 800 22 nd St. NW, Washington, D.C. 20052 2 Department of Electrical and Computer Engineering, Te George Washington University, 800 22 nd St. NW, Washington, D.C. 20052 3 Raith International Applications Center, Raith America, Inc., 300 Jordan Rd., Troy, NY 12180 4 Raith GMBH, Konrad-Adenauer-Allee 8, 44263, Dortmund, Germany *chrisbrantner@email.gwu.edu Abstract: A reverse engineering approach to scanning electron microscope (SEM) imaging of neuronal architecture provides large- area maps of a sample, which links the function of a cell with its location in a tissue. The Chipscanner’s laser interferometer stage and field-of-view mapping allow high-resolution SEM images to be stitched together. This workflow produces accurate, high-resolution maps of tissue over biologically relevant length scales in reasonable time frames. A 2.5 × 1.8 mm mouse spinal cord resin section was imaged in less than 24 hours. This is the most accurate, large-area map of neuronal tissue directly acquired by an SEM. Introduction An inherent limitation of traditional high-resolution electron microscopy (EM) approaches is the limited feld- of-view (FOV), which makes it difcult to relate an object of interest to the overall context of the sample in serial block-face scanning electron microscopy (SEM) [1,2] and automated tape-collecting ultramicrotome SEM [1,3]. Determining such relationships is pivotal for structure-function relationship assignments in the feld of neuroscience. Large-area EM image registrations with seamless junctions between individual images are important in biology to visualize the entire cross section of a tissue or organ. Spinal cord imaging. The architecture of a spinal cord is complex, and an examination of its cellular structures can only be understood by relating them to the larger cyto-architectural map. The specific location of a neuron in the tissue organization gives valuable information of its function. Thus low-magnification images as well as high-magnification images are required for understanding. EM is the “gold standard” for resolution and contrast in samples, yet the FOV and area that can be imaged is relatively small compared to the area of the organized tissue. This small area of a sample that can be imaged in the EM often does not contain landmarks for “knowing” the location of a cell in relation to the entirety of the tissue or organ. In this article a reverse engineering approach for imaging a spinal cord is proposed: the Chipscanner (Raith GmbH) achieves high-accuracy stitching of adjacent high-resolution SEM images covering large areas of tissue. Te combination of high-resolution SEM imaging, laser interferometer stage positioning, and FOV mapping used in this study, produced the most accurate large-area, high-resolution map of spinal cord tissue directly acquired by an SEM instrument reported to date. Te use of a reverse engineering approach to the problem of mapping neurons and their subcellular domains could lead to a better understanding of neuronal architecture in the present context of the BRAIN Initiative in the USA (https://www. whitehouse.gov/share/brain-initiative) and the Human Brain Project in Europe (https://www.humanbrainproject.eu). Microelectronics. With proven accuracy for 22 nm node technologies, the Chipscanner’s high-resolution SEM image Figure 1: Schematic diagram of the Chipscanner chamber. As with single-beam SEM, the Chipscanner offers a variety of electron detectors and multiple-beam energies. (a) Primary electron beam. (b) Everhart- Thornley SE detector. (c) Post-lens angular selective backscatter electron detector (AsB). (d) In-lens detector for secondary and/or energy-selective backscatter electrons (EsB). (e) Sample with varying height. (f) Height- sensing apparatus that uses a laser beam to measure the sample height, keeping the sample in focus over large areas. (g) Laser interferometer stage that provides precise sample motion down to single nanometers. (h) Final polepiece of the SEM. Downloaded from https://www.cambridge.org/core. IP address: 54.163.42.124, on 15 Jun 2020 at 09:04:06, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1551929516000730