Large Volume Electron Microscopy and Neural Microcircuit Analysis

Abstract

One recent technical innovation in neuroscience is microcircuit analysis using three-dimensional reconstructions of neural elements with a large volume Electron microscopy (EM) data set. Large-scale data sets are acquired with newly-developed electron microscope systems such as automated tape-collecting ultramicrotomy (ATUM) with scanning EM (SEM), serial block-face EM (SBEM) and focused ion beam-SEM (FIB-SEM). Currently, projects are also underway to develop computer applications for the registration and segmentation of the serially-captured electron micrographs that are suitable for analyzing large volume EM data sets thoroughly and efficiently. The analysis of large volume data sets can bring innovative research results. These recently available techniques promote our understanding of the functional architecture of the brain.

Keywords: ATUM; FIB-SEM; SBEM; carbon nanotube; connectome; segmentation; synapse; volume electron microscopy.

Figure 1

Pipeline of the automated tape-collecting ultramicrotomy (ATUM)-scanning electron microscopy (SEM) method. The pathway of a Kapton tape (orange color) in the ATUM (Boeckeler Instruments, Inc., Tucson, AZ, USA) is shown in the upper left panel. Sections on tape strings adhered on 4-inch wafer is shown in the upper right. The carbon nanotube (CNT) tape strips in the wafer appear in black, which is a color of a conductive double-sided adhesive tape. Serial sections (gray color) are irregularly clustered on the tape. Copper foil tape between the CNT tape was used to electrically ground the CNT layer to the wafer to secure an escape route for incident electrons. The electron micrographs in lower panels are taken from an ultrathin section of the brain tissue on the CNT tape. The left micrograph was taken with a backscattered electron (BSE) detector of field emission SEM (FE-SEM), Sigma (Carl Zeiss Microscopy GmbH, Oberkochen, Germany) and a large area imaging apparatus for SEM, Atlas 5 (Fibics incorporated, Ottawa, ON, Canada), while middle and right panels were taken with an acceleration voltage 1.5 keV, OnPoint BSE detector (Gatan Inc., Pleasanton, CA, USA) in an FE-SEM, GeminiSEM 300 (Carl Zeiss Microscopy GmbH, Oberkochen, Germany). Adapted from Kubota and Kawaguchi (2018) and Kubota et al. (2018).

Figure 2

Analysis of projected depth of incident electrons. (A–K) Upper panels indicate electron micrographs of brain tissue obtained at various acceleration voltages on an open reel tape. Middle panels show Monte Carlo simulation analysis illustrating potential trajectories of primary and BSEs. (L) Depth in the section that was reached by BSEs as a function of acceleration voltage. Adapted from Kubota and Kawaguchi (2018) and Kubota et al. (2018).

Figure 3

Electron micrographs of rat cerebral cortex. (A) Ultrastructure of rat cerebral cortex. The peripheral portion of the cell body is at the center. The electron micrograph was captured using an acceleration voltage 1.5 keV, dwell time 3 μs/pixel, BSE detector, FE-SEM, Regulus 8240 (Hitachi High-Technologies Corp., Tokyo, Japan). (B) Spine synapse. An enlarged image in the upper left rectangle in (A). Synaptic vesicles and cleft are clearly observed. (C) Somatic synapse. Enlarged image in the lower left rectangle in (A). Synaptic vesicles and clefts are clearly observed. Adapted from Kubota and Kawaguchi (2018) and Kubota et al. (2018).

Figure 4

Procedures and examples of volume EM data set obtained with focused ion beam (FIB)-SEM (FIB-SEM) at 52–54° angle between two beams. (A) Spatial arrangement of FIB, SEM and a tissue block with a mitochondrion. Green line at one side of the block indicates the surface of the tissue block section for light microscopy. “z” is a depth of the imaged volume and “ybs” is y length of the block surface of the image field. (B) An initial part to mill the mitochondrion by FIB. (C) Towards the end of milling for the mitochondrion. (D–F) Captured images of the mitochondrion on the fresh block surface at each milling step. The mitochondrion location gradually deviates in serial SEM images. “x” is × length of image field and “yp” is y length of the captured image. (G) View of the mitochondrion in yz plane. (H) Orthogonal yz view of stacked captured serial images. (I) Orthogonal yz view of stacked scaled serial images. “ysc” is y length of the scaled image, which equals to the “ybs.” (J) Orthogonal yz view of aligned scaled serial images. (K) An original electron micrograph, which is the original image of the first section among 600 serial images of rat frontal cortex captured with FIB-SEM (Helios G4, Thermo Fisher Scientific, Waltham, MT, USA) at 2.29 nm/pixel and 7 nm z-step. The scale bar is for horizontal axis. (L) A scaled electron micrograph of the image shown in (K). (M) Orthogonal yz view of aligned scaled serial images. Please note that the diagram in (J) showing the theoretical orthogonal view of a lozenge, which resembles the orthogonal yz view. (N) Orthogonal xz view of aligned scaled serial images. The scale bar in (M) applies to (L–N), and represents a vertical axis in (K). Modified from Kubota (2015).