Correlative cryo-structured illumination fluorescence microscopy and soft X-ray tomography elucidates reovirus intracellular release pathway
Kounatidis I., Stanifer M., Phillips M., Paul-Gilloteaux P., Helligenstein X., Wang H., Okolo C., Fish T., Spink M., Stuart D., Davis I., Boulant S., Grimes J., Dobbie I., Harkiolaki M.
Abstract Imaging of biological matter across resolution scales presents the challenge of preserving the direct and unambiguous correlation of subject features from the macroscopic to the microscopic level. We present here a correlative imaging platform developed specifically for imaging cells in 3D, under cryogenic conditions. Rapid cryo-preservation of biological specimens is the current gold standard in sample preparation for ultrastructural analysis in X-ray imaging. However, cryogenic fluorescence localisation methods are by and large 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 cryo-preserved states using super-resolution structured illumination microscopy (SIM) in conjunction with soft X-ray tomography (SXT). The power of this new approach is demonstrated by studying the process of reovirus release from intracellular vesicles during the early stages of infection and identifying novel virus-induced structures.