Reference isotropic 3D electron microscopy data of SARS-COV-2-infected grivet kidney epithelial (Vero) cells at 24 h after exposure

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 because they only visualize a single slice or a relatively small volume of the cell, respectively. We overcame these limitations by imaging whole cells and tissues via the enhanced focused ion beam scanning EM (FIB-SEM) platform at 4-nm voxels with month-long acquisition. We used this approach to generate diverse reference 3D image datasets and hope to create a reference library to explore comprehensive quantification of whole cells and all their constituents. These open-access data offer a foundation upon which to nucleate a new volume of high-resolution whole-cell EM images and subsequent analyses, a new paradigm that invites biologists to explore and pose questions with a fresh perspective.SARS-CoV-2-infected grivet kidney epithelial (Vero) cells at the 24-h time point show a high rate of infection with visible convoluted membranes, double-membrane vesicles, vesicle packets, and virus particles.<br><br>Sample: SARS-COV-2-infected grivet kidney epithelial (Vero) cells at the 24-h time point<br><br>Protocol: CCL-81 cells were seeded in a T75 flask at 80% confluency and infected with SARS-CoV-2, at a MOI of 0.01. After 24 hours, the cells were collected from flasks and preserved in 2.5% Glutaraldehyde (E.M. Sciences, Warrington, PA), in Millonig’s Sodium Phosphate Buffer (Tousimis Research, Rockville, MD). After fixation was complete, the cells were washed repeatedly in Millonig’s Buffer, and incubated for two hours in 1.0% Osmium Tetroxide, in the same buffer. Following rinsing steps in Ultrapure Water and en bloc staining with 2.0% Uranyl Acetate, the samples were dehydrated in a series of graded ethanols, and infiltrated and embedded in Spurr’s plastic resin (E.M. Sciences).<br><br>Contributions: Sample provided by John Bernbaum and Jens H. Kuhn ( NIH/NIAID/DCR/Integrated Research Facility at Fort Detrick), prepared for imaging by Song Pang (HHMI/Janelia), imaging by Song Pang (HHMI/Janelia), and post-processing by C. Shan Xu (HHMI/Janelia)<br><br>Dataset ID: jrc_ccl81-covid-1Final voxel size (nm): 4.00 x 4.00 x 4.00 (X, Y, Z)Dimensions (µm): 15 x 20 x 25.8 (X, Y, Z)Acquisition date: 2020-09-10Dataset URL: s3://janelia-cosem-datasets/jrc_ccl81-covid-1/jrc_ccl81-covid-1.zarr/recon-1/em/Visualization Website: https://openorganelle.janelia.org/datasets/jrc_ccl81-covid-1<br>

阐明细胞架构是认知生物学的核心前提。电子显微镜（Electron Microscopy, EM）可凭借纳米级分辨率独特地实现细胞结构的可视化。然而传统方法存在局限性：例如超薄切片EM仅能可视化单个细胞切片，而电子显微镜断层扫描则仅能获取细胞相对狭小的体积范围。我们通过搭载增强型聚焦离子束扫描电子显微镜（Focused Ion Beam Scanning Electron Microscopy, FIB-SEM）的成像平台，以4纳米体素（voxel）的分辨率开展为期一个月的全细胞及组织成像工作，成功突破了上述局限。本研究利用该方法构建了多套标准化三维图像数据集，并期望以此搭建参考库，用于开展全细胞及其所有组分的全面定量分析研究。这批开放获取的数据为海量高分辨率全细胞EM图像及后续分析研究的孕育提供了核心基础，这一全新范式将助力生物学家以全新视角开展探索与科学问题构建。感染严重急性呼吸综合征冠状病毒2型（Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2）的绿猴肾上皮（Vero）细胞在感染后24小时时，感染率较高，且可观测到卷曲膜、双膜囊泡、囊泡簇及病毒颗粒等典型结构。

样本：感染SARS-CoV-2的绿猴肾上皮（Vero）细胞，采集于感染后24小时时间点

实验方案：将CCL-81细胞以80%汇合度接种于T75培养瓶中，以感染复数（Multiplicity of Infection, MOI）为0.01的剂量感染SARS-CoV-2。感染24小时后，收集培养瓶中的细胞，使用含2.5%戊二醛的Millonig磷酸钠缓冲液（购自E.M. Sciences公司，沃灵顿，宾夕法尼亚州）进行固定。固定完成后，用Millonig缓冲液反复冲洗细胞，随后在相同缓冲液中用1.0%四氧化锇孵育2小时。经超纯水冲洗及2.0%乙酸铀酰整体染色后，样品依次经梯度乙醇脱水，再经Spurr's塑料树脂（购自E.M. Sciences公司）渗透包埋。

贡献说明：样本由John Bernbaum与Jens H. Kuhn（美国国立卫生研究院/国家过敏和传染病研究所/DCR/德特里克堡综合研究设施）提供；成像样品制备、成像工作均由Song Pang（霍华德·休斯医学研究所/贾努利亚研究中心，HHMI/Janelia）完成；后期处理由C. Shan Xu（HHMI/Janelia）完成。

数据集编号：jrc_ccl81-covid-1
最终体素尺寸（纳米）：4.00 × 4.00 × 4.00（X、Y、Z轴）
成像尺寸（微米）：15 × 20 × 25.8（X、Y、Z轴）
采集日期：2020年9月10日
数据集链接：s3://janelia-cosem-datasets/jrc_ccl81-covid-1/jrc_ccl81-covid-1.zarr/recon-1/em/
可视化网站：https://openorganelle.janelia.org/datasets/jrc_ccl81-covid-1