DOI: 10.25378/janelia.13114454.v2
title: Isotropic 3D electron microscopy reference data of killer T-Cell attacking cancer cell (jrc_ctl-id8-1)

description: <b>This acquisition is part of the CellMap 2024 Segmentation Challenge</b><b>Challenge DOI:</b> https://doi.org/10.25378/janelia.c.7456966<b>Challenge Website:</b> https://cellmapchallenge.janelia.org/<b>Sample:</b> OT-I mouse cytotoxic T lymphocyte attacking an ID8 cell<b>Sample Description:</b> Understanding cellular architecture is essential for understanding biology. Electron microscopy (EM) uniquely visualizes cellular structure with nanometer resolution. However, traditional methods, such as thin-section EM or EM tomography, have limitations inasmuch as they only visualize a single slice or a relatively small volume of the cell, respectively. Here, we overcome these limitations by long-term imaging whole cells and tissues via the enhanced Focus Ion Beam Scanning Electron Microscopy (FIB-SEM) platform in high resolution mode with month-long acquisition duration. We use this approach to generate reference 3D image data sets at 4-nm isotropic voxels. Together with subsequent segmentation, we hope to create a reference library to explore comprehensive quantification of whole cells and all their constituents, thus addressing questions related to cell identities, cell morphologies, cell-cell interactions, as well as intracellular organelle organization and structure.<b>Cytotoxic T lymphocytes</b> (CTLs) are immune cells that have the capacity to identify and destroy virus-infected or cancerous cells. CTLs are highly motile and patrol tissues in search of these targets. Upon encountering a target, CTLs pause and undergo a dramatic cytoskeletal rearrangement to form an organized intercellular interface called the immunological synapse (IS). Receptor-mediated adhesion and signaling at the IS directs the polarized release of toxic proteins housed in specialized secretory lysosomes called lytic granules (LGs), which results in target cell death. Due to the important role of CTLs in anti-viral and anti-cancer immunity, the IS has been the subject of intense scrutiny. Previous single slice TEM images of CTL:target conjugates have revealed interesting features of the IS, but do not represent the full scope of this dynamic, three-dimensional structure. We here present a 1.4-TB 3D data set (16bit) of a mature murine CTL engaging an ID8 ovarian cancer cell. The isotropic high-resolution information of FIB-SEM imaging provides a unique and complete map of the complex membrane topology at the interface between T cell and target. As such, the stereotypical CTL “cupping” of the target cell at the IS, the variety of features across this interface including membrane interdigitation, flat membrane apposition, filopodia of the target cell trapped between the two cells, and the polarization of the centrosome toward the cancer cell, etc. The unique ability of enhanced FIB-SEM to image whole cells and tissues at 4-nm isotropic voxels over large volumes makes it an ideal tool to map <i>in toto</i> the 3D ultrastructural relationship in living systems.<br><br><b>Protocol</b>: High pressure freezing, freeze-substitution resin embedding with 2% OsO4 0.1% UA 3% H2O in acetone; resin embedding in Eponate 12.<b>Contributions</b>: Sample provided by Alex Ritter (Genentech), prepared for imaging by Gleb Shtengel (HHMI/Janelia), with imaging and post-processing by C. Shan Xu (HHMI/Janelia).<b>Acquisition ID</b>: jrc_ctl-id8-1<b>Final voxel size (nm)</b>: 4.00 x 4.00 x 3.48 (X, Y, Z)<b>Dimensions (µm)</b>: 74 x 13 x 42 (X, Y, Z)<b>Imaging start date</b>: 2020-02-05<b>Imaging duration (days)</b>: 29<b>Landing energy (eV)</b>: 700<b>Imaging current (nA)</b>: .25<b>Scanning speed (MHz)</b>: .2<b>Dataset URL</b>: s3://janelia-cosem-datasets/jrc_ctl-id8-1/jrc_ctl-id8-1.zarr/recon-1/em/<b>Visualization Website</b>: https://openorganelle.janelia.org/datasets/jrc_ctl-id8-1<br><br><b>Publication</b>: Xu et al., 2021<br>