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本数据集隶属于CellMap 2024图像分割挑战赛（CellMap 2024 Segmentation Challenge），挑战赛DOI：https://doi.org/10.25378/janelia.c.7456966，挑战赛官网：https://cellmapchallenge.janelia.org/ 样本：攻击ID8细胞的OT-I小鼠细胞毒性T淋巴细胞 样本说明：解析细胞架构是理解生命科学的核心基础。电子显微镜（Electron Microscopy, EM）可实现纳米级分辨率的细胞结构可视化，具备独特技术优势。然而传统电子显微镜成像方法，如超薄切片电子显微镜或电子断层扫描技术，分别存在仅能获取单切片图像或仅覆盖较小细胞体积的局限性。本研究通过搭载增强型聚焦离子束扫描电子显微镜（Focus Ion Beam Scanning Electron Microscopy, FIB-SEM）平台，以高分辨率模式开展长达一月的全细胞与组织长期成像，成功克服了上述局限。我们采用该方法生成了各向同性体素分辨率为4纳米的参考三维图像数据集。结合后续图像分割工作，我们旨在构建参考数据库，以实现对全细胞及其所有组成成分的全面定量分析，从而解决与细胞身份、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科学问题。 细胞毒性T淋巴细胞（Cytotoxic T Lymphocytes, CTLs）是一类可识别并杀伤病毒感染或癌变细胞的免疫细胞。CTLs具有高度运动能力，可在组织中巡逻以搜寻靶标。当识别到靶细胞后，CTLs会停止移动，并经历剧烈的细胞骨架重排，形成名为免疫突触（Immunological Synapse, IS）的结构化细胞间界面。免疫突触处的受体介导黏附与信号传导，可引导储存于特异性分泌溶酶体（即溶细胞颗粒，Lytic Granules, LGs）中的毒性蛋白极化释放，最终导致靶细胞死亡。鉴于CTLs在抗病毒与抗肿瘤免疫中的关键作用，免疫突触一直是国际研究的热点领域。此前针对CTL-靶细胞结合物的单切片透射电子显微镜（Transmission Electron Microscopy, TEM）图像已揭示了免疫突触的诸多有趣特征，但无法完整呈现这一动态三维结构。 本研究公开了一份容量为1.4 TB的16位三维数据集，对应成熟小鼠CTL结合ID8卵巢癌细胞的成像结果。FIB-SEM成像所获得的各向同性高分辨率信息，可完整呈现T细胞与靶细胞界面处复杂的膜拓扑结构，具备独特的研究价值。具体而言，该数据集包含CTL在免疫突触处对靶细胞的典型“杯状”包裹形态、界面处的多种特征（如膜相互嵌合、平坦膜贴合、夹在两细胞间的靶细胞丝状伪足，以及中心体向癌细胞的极化现象等）。增强型FIB-SEM可在大体积范围内以4纳米各向同性体素分辨率对全细胞与组织进行成像，这一独特能力使其成为在活体系统中全面绘制三维超微结构关系的理想工具。 实验方案：高压冷冻、以丙酮中含2%四氧化锇（OsO4）、0.1%乙酸铀（UA）、3%水的溶液进行冷冻替代树脂包埋，随后使用Eponate 12树脂进行包埋。 贡献说明：样本由基因泰克（Genentech）的Alex Ritter提供，霍华德·休斯医学研究所/贾内利亚研究园区（HHMI/Janelia）的Gleb Shtengel完成成像前样品制备，HHMI/Janelia的C. Shan Xu负责成像与后期处理。 采集ID：jrc_ctl-id8-1 最终体素尺寸（纳米）：4.00 × 4.00 × 3.48（X、Y、Z轴） 成像维度（微米）：74 × 13 × 42（X、Y、Z轴） 成像开始日期：2020-02-05 成像时长（天）：29 加速电压（电子伏特）：700 成像电流（纳安）：0.25 扫描速度（兆赫兹）：0.2 数据集URL：s3://janelia-cosem-datasets/jrc_ctl-id8-1/jrc_ctl-id8-1.zarr/recon-1/em/ 可视化官网：https://openorganelle.janelia.org/datasets/jrc_ctl-id8-1 相关文献：Xu et al., 2021