3D Correlative Cryo-Structured Illumination Fluorescence and Soft X-ray Microscopy Elucidates Reovirus Intracellular Release Pathway

• Published in: Cell Vol. 182; no. 2; pp. 515 - 530.e17
• Main Authors: Kounatidis, Ilias, Stanifer, Megan L., Phillips, Michael A., Paul-Gilloteaux, Perrine, Heiligenstein, Xavier, Wang, Hongchang, Okolo, Chidinma A., Fish, Thomas M., Spink, Matthew C., Stuart, David I., Davis, Ilan, Boulant, Steeve, Grimes, Jonathan M., Dobbie, Ian M., Harkiolaki, Maria
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 23.07.2020; Elsevier; Cell Press
• Subjects: Bioengineering; Cell Line, Tumor; CLXT; computed tomography; Computer Science; Computer Vision and Pattern Recognition; correlative imaging; Cryoelectron Microscopy - instrumentation; Cryoelectron Microscopy - methods; Endosomes - metabolism; Endosomes - virology; fluorescence; fluorescence microscopy; Fluorescent Dyes - chemistry; Humans; ice; image analysis; Imaging; Imaging, Three-Dimensional; Life Sciences; lighting; Microscopy, Fluorescence; Reoviridae; Reoviridae - chemistry; Reoviridae - physiology; reovirus biology; structured illumination microscopy; Virus Release - physiology; X-radiation; X-ray tomography; X-ray tomography; reovirus biology; CLXT; structured illumination microscopy; correlative imaging
• ISSN: 0092-8674; 1097-4172; 1097-4172
• DOI: 10.1016/j.cell.2020.05.051

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. [Display omitted] •Correlative cryo-imaging used to study biological processes in vitrified cells•Reovirus intracellular release mechanism is elucidated in 3D with combined cryo-imaging•Reovirus core particles start modifying carrier endosomes at 1 h after infection•Correlation of X-ray and fluorescence cryo-imaging requires no data deformation Kounatidis et al. describe a correlative microscopy platform that combines cryo-structured illumination fluorescence imaging with soft X-ray absorption imaging on the same sample allowing direct data correlation in 3D. The methods are used to track the intracellular progress of reovirus in human cells in the early stages of infection.