Isotropic 3D electron microscopy reference data of killer T-Cell attacking cancer cell (jrc_ctl-id8-1)

This acquisition is part of the CellMap 2024 Segmentation ChallengeChallenge DOI: https://doi.org/10.25378/janelia.c.7456966Challenge Website: https://cellmapchallenge.janelia.org/Sample: OT-I mouse cytotoxic T lymphocyte attacking an ID8 cellSample Description: 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.Cytotoxic T lymphocytes (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 in toto the 3D ultrastructural relationship in living systems.Protocol: High pressure freezing, freeze-substitution resin embedding with 2% OsO4 0.1% UA 3% H2O in acetone; resin embedding in Eponate 12.Contributions: 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).Acquisition ID: jrc_ctl-id8-1Final voxel size (nm): 4.00 x 4.00 x 3.48 (X, Y, Z)Dimensions (µm): 74 x 13 x 42 (X, Y, Z)Imaging start date: 2020-02-05Imaging duration (days): 29Landing energy (eV): 700Imaging current (nA): .25Scanning speed (MHz): .2Dataset URL: s3://janelia-cosem-datasets/jrc_ctl-id8-1/jrc_ctl-id8-1.zarr/recon-1/em/Visualization Website: https://openorganelle.janelia.org/datasets/jrc_ctl-id8-1Publication: Xu et al., 2021

本采集数据属于2024年CellMap分割挑战赛的一部分，挑战赛DOI：https://doi.org/10.25378/janelia.c.7456966，挑战赛网站：https://cellmapchallenge.janelia.org。样本：OT-I小鼠细胞毒性T淋巴细胞攻击ID8细胞。样本描述：细胞结构的理解对于生物学的研究至关重要。电子显微镜（EM）以其纳米级的分辨率独特地可视化了细胞结构。然而，传统的薄切片EM或EM断层扫描等方法在可视化单个切片或相对较小的细胞体积方面存在局限性。在本研究中，我们通过增强聚焦离子束扫描电子显微镜（FIB-SEM）平台在高分辨率模式下进行长期全细胞和组织的成像，克服了这些局限性，成像时长达数月。我们采用此方法生成4纳米等距体素的三维参考图像数据集。结合后续的分割，我们期望创建一个参考库，以探索对整个细胞及其所有组成部分的全面量化，从而解答与细胞身份、细胞形态、细胞间相互作用，以及细胞内细胞器组织与结构相关的问题。细胞毒性T淋巴细胞（CTLs）是一种具有识别和破坏病毒感染细胞或癌细胞能力的免疫细胞。CTLs具有高度的活动性，并在组织中巡逻以寻找这些靶标。当遇到靶标时，CTLs会暂停并进行显著的细胞骨架重排，形成称为免疫突触（IS）的有组织的细胞间界面。在IS处的受体介导的粘附和信号传导指导着毒蛋白的极化释放，这些毒蛋白位于特化的分泌溶酶体中，称为溶酶体（LGs），从而导致靶细胞死亡。由于CTLs在抗病毒和抗癌免疫中的重要作用，免疫突触一直是研究的焦点。先前CTL：靶标共轭的单切片透射电镜（TEM）图像揭示了免疫突触的有趣特征，但并未代表这一动态的三维结构的全貌。在此，我们呈现了一个1.4TB的成熟小鼠CTL与ID8卵巢癌细胞相互作用的3D数据集（16位）。FIB-SEM成像的等距高分辨率信息提供了T细胞与靶细胞界面处复杂膜拓扑结构的独特且完整的映射。因此，典型的CTL在免疫突触处对靶细胞的“捧杯”状，以及该界面上的各种特征，包括膜相互穿插、平坦膜附着、靶细胞伪足被夹在两个细胞之间，以及中心体向癌细胞极化的现象等。增强FIB-SEM在4纳米等距体素下对整个细胞和组织进行成像的独特能力，使其成为映射活系统中三维超微结构关系的理想工具。实验方法：高压冷冻，冻替换树脂嵌入，使用2%的OsO4、0.1%的UA和3%的H2O在丙酮中；在Eponate 12中进行树脂嵌入。贡献：样本由Alex Ritter（基因泰克）提供，由Gleb Shtengel（HHMI/Janelia）准备用于成像，由C. Shan Xu（HHMI/Janelia）进行成像和后期处理。采集ID：jrc_ctl-id8-1。最终体素大小（nm）：4.00 x 4.00 x 3.48（X，Y，Z）。尺寸（µm）：74 x 13 x 42（X，Y，Z）。成像开始日期：2020-02-05。成像时长（天）：29。着陆能量（eV）：700。成像电流（nA）：.25。扫描速度（MHz）：.2。数据集URL：s3://janelia-cosem-datasets/jrc_ctl-id8-1/jrc_ctl-id8-1.zarr/recon-1/em。可视化网站：https://openorganelle.janelia.org/datasets/jrc_ctl-id8-1。出版物：Xu et al.，2021。