Integrated multimodality microscope for accurate and efficient target-guided cryo-lamellae preparation
Weixing Li, Jing Lu, Ke Xiao, Maoge Zhou, Yuanyuan Li, Xiang Zhang, Zhixun Li, Lusheng Gu, Xiaojun Xu, Qiang Guo, Tao Xu & Wei Ji
Abstract
Cryo-electron tomography (cryo-ET) is a revolutionary technique for resolving the structure of subcellular organelles and macromolecular complexes in their cellular context. However, the application of the cryo-ET is hampered by the sample preparation step. Performing cryo-focused ion beam milling at an arbitrary position on the sample is inefficient, and the target of interest is not guaranteed to be preserved when thinning the cell from several micrometers to less than 300?nm thick. Here, we report a cryogenic correlated light, ion and electron microscopy (cryo-CLIEM) technique that is capable of preparing cryo-lamellae under the guidance of three-dimensional confocal imaging. Moreover, we demonstrate a workflow to preselect and preserve nanoscale target regions inside the finished cryo-lamellae. By successfully preparing cryo-lamellae that contain a single centriole or contact sites between subcellular organelles, we show that this approach is generally applicable, and shall help in innovating more applications of cryo-ET.
附件下载：
Integrated multimodality microscope for accurate and efficient target-guided cryo-lamellae preparation, Nat Methods, 16 Jan 2023
Nat Methods, 16 January, 2023, DOI：https://doi.org/10.1038/s41592-022-01749-z
Integrated multimodality microscope for accurate and efficient target-guided cryo-lamellae preparation
Weixing Li, Jing Lu, Ke Xiao, Maoge Zhou, Yuanyuan Li, Xiang Zhang, Zhixun Li, Lusheng Gu, Xiaojun Xu, Qiang Guo, Tao Xu & Wei Ji
Abstract
Cryo-electron tomography (cryo-ET) is a revolutionary technique for resolving the structure of subcellular organelles and macromolecular complexes in their cellular context. However, the application of the cryo-ET is hampered by the sample preparation step. Performing cryo-focused ion beam milling at an arbitrary position on the sample is inefficient, and the target of interest is not guaranteed to be preserved when thinning the cell from several micrometers to less than 300?nm thick. Here, we report a cryogenic correlated light, ion and electron microscopy (cryo-CLIEM) technique that is capable of preparing cryo-lamellae under the guidance of three-dimensional confocal imaging. Moreover, we demonstrate a workflow to preselect and preserve nanoscale target regions inside the finished cryo-lamellae. By successfully preparing cryo-lamellae that contain a single centriole or contact sites between subcellular organelles, we show that this approach is generally applicable, and shall help in innovating more applications of cryo-ET.
文章链接：https://www.nature.com/articles/s41592-022-01749-z
相关报道：http://www.ibp.cas.cn/kyjz/zxdt/202301/t20230113_6599093.html