Summary 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.

We derive the recent growth history of the Fornax Cluster, in particular the recent infall of the giant elliptical galaxy NGC 1404. We show, using a simple cluster minor merger simulation tailored to Fornax and NGC 1404, that a second or more likely third encounter between the two reproduces all main merger features observed in both objects; we firmly exclude a first infall scenario. Our simulations reveal a consistent picture: NGC 1404 passed by NGC 1399 about 1.1 -1.3 Gyrs ago from the NE to the SW and is now almost at the point of its next encounter from the S. This scenario explains the sloshing patterns observed in Fornax -a prominent northern cold front and an inner southern cold front. This scenario also explains the truncated atmosphere, the gas stripping radius of NGC 1404, and its faint gas tail. Independent of the exact history, we can make a number of predictions. A detached bow shock south of NGC 1404 should exist which is a remnant of the galaxy's previous infall at a distance from NGC 1404 between 450 -750 kpc with an estimated Mach number between 1.3 and 1.5. The wake of NGC 1404 also lies S of the galaxy with enhanced turbulence and a slight enhancement in metallicity compared to the undisturbed regions of the cluster. SW of NGC 1404, there is likely evidence of old turbulence originating from the previous infall. No scenario predicts enhanced turbulence outside of the cold front north west of the cluster center.

We show that there is a new class of gas tails -slingshot tails -which form as a subhalo (i.e. a subcluster or early-type cluster galaxy) moves away from the cluster center towards the apocenter of its orbit. These tails can point perpendicular or even opposite to the subhalo direction of motion, not tracing the recent orbital path. Thus, the observed tail direction can be misleading, and we caution against naive conclusions regarding the subhalo's direction of motion based on the tail direction. A head-tail morphology of a galaxy's or subcluster's gaseous atmosphere is usually attributed to ram pressure stripping and the widely applied conclusion is that gas stripped tail traces the most recent orbit. However, during the slingshot tail stage, the subhalo is not being ram pressure stripped (RPS) and the tail is shaped by tidal forces more than just the ram pressure. Thus, applying a classic RPS scenario to a slingshot tail leads not only to an incorrect conclusion regarding the direction of motion, but also to incorrect conclusions in regard to the subhalo velocity, expected locations of shear flows, instabilities and mixing. We describe the genesis and morphology of slingshot tails using data from binary cluster merger simulations, discuss their observable features and how to distinguish them from classic RPS tails. We identify three examples from the literature that are not RPS tails but slingshot tails and discuss other potential candidates.

This protocol describes sample preparation strategies for correlative 3D cryo-SIM and cryo soft X-ray tomography. In addition, the authors provide a direct comparison and recommendations regarding the selection and use of fiducials for 3D correlation. TWEET A new protocol for sample preparation and fiducial selection for correlative cryo-SIM and cryo-soft X-ray tomography. #CLXT #cryoimaging #cellstructure #correlativeimaging COVER TEASER Correlative cryo-SIM and cryo-soft X-ray tomographyAbstract 3D correlative microscopy methods have revolutionised biomedical research allowing the acquisition of multi-dimensional information to gain an in-depth understanding of biological systems. With the advent of relevant cryo-preservation methods, correlative imaging of cryogenically preserved samples has led to nanometre resolution imaging (2-50 nm) under harsh imaging regimes such as electron and soft X-ray tomography. These methods have now been combined with conventional and super resolution fluorescence imaging at cryogenic temperatures to augment information content from a given sample resulting in the immediate requirement for protocols that facilitate hassle-free unambiguous cross correlation between microscopes. We present here sample preparation strategies and a direct comparison of different working fiducialisation regimes that facilitate 3D correlation of cryo-structured illumination microscopy and cryo-soft X-ray tomography. Our protocol has been tested at two synchrotron beamlines (B24 at Diamond Light Source in the UK and BL09 Mistral at ALBA in Spain) and has led to the development of a decision aid that facilitates experimental design with the strategic use of markers based on project requirements. This protocol takes between 1.5 hours and 3.5 days to complete, depending on the cell populations used (adherent cells may require several days to grow on sample carriers).