Near-isotropic, reconstructed volume electron microscopy (FIB-SEM) of human airway (jrc_hum-airway-14953vc)

Sample: Human Airway Epithelium, from immortalised basal stem cell progenitors, differentiated using Air Liquid Interphase (ALI) cultures
Sample Description: This sample captures the cellular diversity of the human bronchial airway epithelium, the superficial layer of the respiratory tract whose major function is to provide a physical, biochemical and immunological barrier from the infection of viruses, bacteria and environmental pollutants inhaled through breathing.
Protocol: Human multiciliated cells cultures differentiated for 28 days were fixed with Karnovsky’s fixative in 0.1M sodium cacodylate buffer (SCB, pH 7.4) with 0.05% malachite green oxalate salt and processed largely as previously described (Liu et al., 2020; Morone et al., 2024). Briefly, specimens were post-fixed with the osmium-thiocarbohydrazide-osmium (OTO) method, with incubations in 1% osmium tetroxide in SCB for 1.5 hrs at RT, 2.5% potassium ferrocyanide in SCB for 1.5 hrs at RT, aqueous 1% thiocarbohydrazide solution for 45 mins at 40°C, and then with a 2% osmium tetroxide aqueous solution for 1.5 hrs at RT, followed by en-bloc staining with aqueous 1% uranyl acetate solution overnight at 4°C. Samples were subsequently transferred in lead aspartate solution (pH 5.0) for 2 hrs at 50°C. After dehydration in ethanol/acetonitrile series the sample was embedded in TAAB E202(/1) Araldite/TAAB 812 Epon mixture Resin.
Contributions: Region of Interest (ROI) and biology description provided by Vito Mennella (University of Cambridge), sample provided by Vito Mennella, Nobu Morone and Sophie Breugesem (University of Cambridge), prepared for imaging by Zhiyuan Lu (HHMI/Janelia), with imaging by Wei Qiu (HHMI/Janelia) and Christopher Bleck (HHMI/Janelia), post-processing by Eric Trautman, Michael Innerberger and Stephan Preibisch (HHMI/Janelia).
Acquisition ID: jrc_hum-airway-14953vc
Voxel size (nm): 8 x 8 x 8 (x, y, z)
Data dimensions (µm): 212.94 x 103.02 x 120.74 (x, y, z)
Imaging start date: 2023-11-21
Imaging duration (days): 32
Dataset URL: https://data.janelia.org/gV96S
Visualization Website: https://openorganelle.janelia.org/datasets/jrc_hum-airway-14953vc

Publication: Xu et al., 2017; Vijayakumaran, et al., 2024

样本：源自永生化基底层干细胞祖细胞的气道上皮细胞，采用气液界面（ALI）培养分化而成。
样本描述：本样本捕捉了人类支气管气道上皮细胞的细胞多样性，该上皮细胞为呼吸道的表层，其主要功能是为抵御通过呼吸吸入的病毒、细菌和环境污染物的感染，提供物理、生化及免疫屏障。
实验方法：对分化28天的人多纤毛细胞进行固定，采用Karnovsky固定液在0.1M钠草酸盐缓冲液（SCB，pH 7.4）中，加入0.05%孔雀石绿草酸盐，并主要依照先前描述的方法进行处理（Liu等，2020；Morone等，2024）。简而言之，样本经铀-硫代碳酰胺-铀（OTO）方法进行后固定，在室温下用SCB中的1%四氧化铀溶液孵育1.5小时，2.5%铁氰化钾在SCB中孵育1.5小时，40°C下用水溶液中的1%硫代碳酰胺溶液孵育45分钟，随后用室温下的2%四氧化铀水溶液孵育1.5小时，随后在4°C下过夜用1%醋酸铀水溶液进行整体染色。样本随后转移至铅天冬氨酸溶液（pH 5.0）中，50°C下孵育2小时。在乙醇/乙腈系列脱水后，样本被包埋在TAAB E202(/1) Araldite/TAAB 812 Epon混合树脂中。
贡献：兴趣区域（ROI）及生物学描述由Vito Mennella（剑桥大学）提供，样本由Vito Mennella、Nobu Morone和Sophie Breugesem（剑桥大学）提供，成像前由Zhiyuan Lu（HHMI/Janelia）准备，成像由Wei Qiu（HHMI/Janelia）和Christopher Bleck（HHMI/Janelia）完成，后期处理由Eric Trautman、Michael Innerberger和Stephan Preibisch（HHMI/Janelia）负责。
采集ID：jrc_hum-airway-14953vc
体素大小（nm）：8 x 8 x 8（x, y, z）
数据维度（µm）：212.94 x 103.02 x 120.74（x, y, z）
成像开始日期：2023-11-21
成像持续时间（天）：32
数据集URL：https://data.janelia.org/gV96S
可视化网站：https://openorganelle.janelia.org/datasets/jrc_hum-airway-14953vc

出版物：Xu等，2017；Vijayakumaran等，2024