Isotropic 3D electron microscopy reference data of COS-7 cell overexpressing ER and mitochondria markers for correlative light and electron microscopy studies (jrc_cos7-11)

Within cells, the spatial compartmentalization of thousands of distinct proteins serves a multitude of diverse biochemical needs. Correlative super-resolution (SR) fluorescence and electron microscopy (EM) can elucidate protein spatial relationships to global ultrastructure, but has suffered from tradeoffs of structure preservation, fluorescence retention, resolution, and field of view. We developed a platform for three-dimensional cryogenic SR and focused ion beam-milled block-face EM across entire vitreously frozen cells. The approach preserves ultrastructure while enabling independent SR and EM workflow optimization. We discovered unexpected protein-ultrastructure relationships in mammalian cells including intranuclear vesicles containing endoplasmic reticulum-associated proteins, web-like adhesions between cultured neurons, and chromatin domains subclassified on the basis of transcriptional activity. Our findings illustrate the value of a comprehensive multimodal view of ultrastructural variability across whole cells.Sample: COS-7 cell overexpressing mEmerald-ER3 and Halo/JF525-TOMM20 (ATCC CRL-1651)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 Melanie Freeman (HHMI/Janelia), prepared for imaging by Gleb Shtengel (HHMI/Janelia), with imaging and post-processing by C. Shan Xu (HHMI/Janelia).Dataset ID: jrc_cos7-11Final voxel size (nm): 8.0 x 8.0 x 8.0 (X, Y, Z)Dimensions (µm): 70 x 8 x 100 (X, Y, Z)Acquisition date: 2018-04-27Dataset URL: s3://janelia-cosem-datasets/jrc_cos7-11/jrc_cos7-11.zarr/recon-1/em/Visualization Website: https://openorganelle.janelia.org/datasets/jrc_cos7-11Publication: Hoffman et al., 2020 (10.1126/science.aaz5357)<br>

细胞内，数千种不同蛋白质的空间区室化可满足多样化的生物化学功能需求。关联式超分辨率（super-resolution, SR）荧光显微镜与电子显微镜（electron microscopy, EM）技术，能够阐明蛋白质与整体细胞超微结构的空间关联，但此前一直面临结构保存率、荧光保留率、分辨率及视场范围之间的权衡取舍问题。我们开发了一套可对完整玻璃化冷冻细胞进行三维低温超分辨率成像与聚焦离子束铣削块面电子显微镜成像的平台。该方案在完整保存细胞超微结构的同时，可实现超分辨率成像与电子显微镜成像流程的独立优化。我们在哺乳动物细胞中发现了此前未被报道的蛋白质-超微结构关联，包括携带内质网相关蛋白的核内囊泡、体外培养神经元间的网状黏附结构，以及基于转录活性进行亚型分类的染色质结构域。本研究结果证实了全细胞超微结构变异的多模态综合观测的重要研究价值。
样本：过表达mEmerald-ER3与Halo/JF525-TOMM20的COS-7细胞（ATCC CRL-1651）
实验方案：采用高压冷冻技术，经含2%四氧化锇（OsO4）、0.1%乙酸铀（UA）与3%水的丙酮溶液进行冷冻替代，随后使用Eponate 12树脂进行包埋
贡献说明：样本由Melanie Freeman（HHMI/Janelia）提供，成像前样本制备由Gleb Shtengel（HHMI/Janelia）完成，成像与后处理工作由C. Shan Xu（HHMI/Janelia）执行
数据集ID：jrc_cos7-11
最终体素尺寸（纳米）：8.0×8.0×8.0（X、Y、Z轴）
成像视野尺寸（微米）：70×8×100（X、Y、Z轴）
数据采集日期：2018-04-27
数据集访问链接：https://data.janelia.org/X2jWw
可视化网站：https://openorganelle.janelia.org/datasets/jrc_cos7-11
发表文献：Hoffman等，2020（DOI: 10.1126/science.aaz5357）