3D Correlative Cryo-Structured Illumination Fluorescence and Soft X-ray Microscopy Elucidates Reovirus Intracellular Release Pathway
3D Correlative Cryo-Structured Illumination Fluorescence and Soft X-ray Microscopy Elucidates Reovirus Intracellular Release Pathway
复制标题
DOI：
10.1016/j.cell.2020.05.051
复制
发表时间：
2020-07-23
期刊：
影响因子：
64.5
通讯作者：
Harkiolaki, Maria
中科院分区：
文献类型：
--
作者：
Kounatidis, Ilias;Stanifer, Megan L.;Harkiolaki, Maria
跨分辨率尺度的生物物质成像面临着从宏观到微观层面保持主题特征直接且明确的相关性的挑战。在这里，我们提出了一个专门为低温条件下使用 X 射线和可见光对细胞进行 3D 成像而开发的相关成像平台。生物标本的快速冷冻保存是当前 X 射线成像超微结构分析样品制备的黄金标准。然而，低温荧光定位方法大多数都受到衍射限制，无法提供匹配的分辨率。我们通过开发一个集成的、用户友好的平台来解决这一技术差距，通过使用超分辨率结构照明显微镜与软 X 射线断层扫描相结合，对玻璃体冰中的细胞进行 3D 相关成像。通过研究感染早期阶段呼肠孤病毒从细胞内囊泡释放的过程并鉴定细胞内病毒诱导的结构，证明了这种方法的威力。
Imaging of biological matter across resolution scales entails the challenge of preserving the direct and unambiguous correlation of subject features from the macroscopic to the microscopic level, Here, we present a correlative imaging platform developed specifically for imaging cells in 3D under cryogenic conditions by using X-rays and visible light. Rapid cryo-preservation of biological specimens is the current gold standard in sample preparation for ultrastructural analysis in X-ray imaging. However, cryogenic fluorescence localization methods are, in their majority, diffraction-limited and fail to deliver matching resolution, We addressed this technological gap by developing an integrated, user-friendly platform for 3D correlative imaging of cells in vitreous ice by using super-resolution structured illumination microscopy in conjunction with soft X-ray tomography. The power of this approach is demonstrated by studying the process of reovirus release from intracellular vesicles during the early stages of infection and identifying intracellular virus-induced structures.