The performance of MISTRAL is reported, the soft X-ray transmission microscopy beamline at the ALBA light source (Barcelona, Spain) which is primarily dedicated to cryo soft X-ray tomography (cryo-SXT) for three-dimensional visualization of whole unstained cells at spatial resolutions down to 30 nm (half pitch). Short acquisition times allowing for high-throughput and correlative microscopy studies have promoted cryo-SXT as an emerging cellular imaging tool for structural cell biologists bridging the gap between optical and electron microscopy. In addition, the beamline offers the possibility of imaging magnetic domains in thin magnetic films that are illustrated here with an example.

Advances in nanoscale magnetism increasingly require characterization tools providing detailed descriptions of magnetic configurations. Magnetic transmission X-ray microscopy produces element specific magnetic domain images with nanometric lateral resolution in films up to ∼100 nm thick. Here we present an imaging method using the angular dependence of magnetic contrast in a series of high resolution transmission X-ray microscopy images to obtain quantitative descriptions of the magnetization (canting angles relative to surface normal and sense). This method is applied to 55–120 nm thick ferromagnetic NdCo5 layers (canting angles between 65° and 22°), and to a NdCo5 film covered with permalloy. Interestingly, permalloy induces a 43° rotation of Co magnetization towards surface normal. Our method allows identifying complex topological defects (merons or ½ skyrmions) in a NdCo5 film that are only partially replicated by the permalloy overlayer. These results open possibilities for the characterization of deeply buried magnetic topological defects, nanostructures and devices.

Imaging techniques in structural cell biology are indispensable to understand cell organization and machinery. In this frame, cryo soft X-ray tomography (cryo-SXT), a synchrotron-based imaging technique, is used to analyze the ultrastructure of intact, cryo-preserved cells at nanometric spatial resolution bridging electron microscopy and visible light fluorescence. With their unique interaction with matter and high penetration depth, X-rays are a very useful and complementary source to obtain both high-resolution and quantitative information. In this review, we are elaborating a typical cryo correlative workflow at the Mistral Beamline at the Alba Synchrotron (Spain) with the goal of providing a cartographic description of the cell by cryo-SXT that illustrates the possibilities this technique brings for specific localization of cellular features, organelle organization, and particular events in specific structural cell biology research.

Thin perpendicular magnetic anisotropy films between two soft ferromagnetic layers have the nuclei for magnetization inversion at the bifurcations of their characteristic stripe domain pattern. The inverted nuclei induce vortex-antivortex pairs in the soft magnetic layers that exhibit a correlated motion extending several μm along the magnetic stripes during magnetization reversal. The sense of motion is completely determined by the topology of the magnetic bifurcations causing vortex-antivortex pairs to propagate in opposite senses depending on their polarities. This is a robust effect that might have practical applications. These findings are based on X-ray microscopy and micromagnetic calculations.

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these techniques, cryo soft X-ray tomography (SXT) allows imaging whole cryo-preserved cells in the water window X-ray energy range (284-543 eV), in which carbon structures have intrinsically higher absorption than water, allowing the 3D reconstruction of the linear absorption coefficient of the material contained in each voxel. Quantitative structural information at the level of whole cells up to 10 µm thick is then achievable this way, with high throughput and spatial resolution down to 25-30 nm half-pitch. Cryo-SXT has proven itself relevant to current biomedical research, providing 3D information on cellular infection processes (virus, bacteria, or parasites), morphological changes due to diseases (such as recessive genetic diseases) and helping us understand drug action at the cellular level, or locating specific structures in the 3D cellular environment. In addition, by taking advantage of the tunable wavelength at synchrotron facilities, spectro-microscopy or its 3D counterpart, spectrotomography, can also be used to image and quantify specific elements in the cell, such as calcium in biomineralization processes. Cryo-SXT provides complementary information to other biological imaging techniques such as electron microscopy, X-ray fluorescence or visible light fluorescence, and is generally used as a partner method for 2D or 3D correlative imaging at cryogenic conditions in order to link function, location, and morphology.

The magnetization reversal of each individual layer in magnetic trilayers (permalloy/NdCo/GdCo) is investigated in detail with x-ray microscopy and micromagnetic calculations. Two sequential inversion mechanisms are identified. First, magnetic vortex-antivortex pairs move along the field direction while inverting the magnetization of magnetic stripes until they are pinned by defects. The vortex-antivortex displacements are reversible within a field interval which allows their controlled motion. Second, as the reversed magnetic field increases, cycloidal domains appear in the permalloy layer as a consequence of the dissociation of vortex-antivortex pairs due to pinning. The field range where magnetic vortices and antivortices are effectively guided by the stripe pattern is of the order of tens of mT for the NiFe layer, as estimated from the stability of cycloid domains in the sample.

Soft X-Ray cryo tomography has become a tool for analyzing the ultrastructure of intact and cryoprepared cells. No other method can provide better 3D resolution of the whole cell organelles preserved close to native state. Nevertheless the tilt angle range from which data can be gathered is limited from -65º to 65º or -70º to 70º depending on the zone plate used. This limitation causes the truncation of the data, represented in reciprocal space as a missing wedge which is an important artifact in the reconstructed tomograms that affects the axial direction. The effect of the missing wedge is very strong on details perpendicular to the optical axis. One way to reduce this artefact is to acquire data from two perpendicular tilt axes, a technique called ''dual axis tomography.'' 1 In the current work we have designed a holder for MISTRAL microscope that facilitate the acquisition of two tilt series of the same area of interest whose tilt axes are mutually perpendicular. Dual Tilt leaded to decrease the missing wedge to a missing pyramid and to obtain a more isotropic axial resolution in the volume helping to visualized structures that were hidden in the single tilt volume due to their orientation with respect to the axis of rotation. The dual axis tilting method increases the visibility of certain features which are hidden by the missing wedge; this makes the resolution achievable more isotropic inside the volume (Fig 1). On the other hand the new holder allows us loading samples in an autogrid ring. The autogrid system protects grids during handling and facilitates the correlative microscopy making possible the analysis of the same samples with different techniques. Correlation of cryo-fluorescence microscopy and cryo-SXT can be used to localize fluorescent proteins tagging specific organelles. Cryo-correlative light and X-ray microscopy (cryo-CLXM) is particularly useful for the study of organelles that are susceptible to chemical fixation artifacts during sample preparation for electron microscopy 2 . We have selected the Hepatitis C virus as a biological model for the study.Chronic hepatitis C virus (HCV) infection causes severe liver disease in millions of humans worldwide. Pathogenesis of HCV infection is strongly driven by a deficient immune response of the host, although intersection of different aspects of the virus life cycle with cellular homeostasis is emerging as an important player in the pathogenesis and progression of the disease.Cryo soft X-ray tomography (cryo-SXT) was performed to investigate the ultrastructural alterations induced by the interference of hepatitis C virus (HCV) replication with cellular homeostasis. Native, whole cell, three-dimensional maps were obtained in HCV replicon-harboring cells and in a surrogate model of HCV infection at 40nm resolution. Tomograms from HCV-replicating cells show blind-ended endoplasmic reticulum (ER) tubules with pseudo spherical extrusions and marked alterations of mitochondrial morphology that correlated topologically with the presence of ER alteratio...

Hepatitis C virus (HCV) is an enveloped RNA virus. One of the hallmarks of HCV infection is a rearrangement of the host cell membranes, known as the `membranous web'. Full-field cryo soft X-ray tomography (cryo-SXT) in the water-window energy range (284–543 eV) was performed on the MISTRAL beamline to investigate, in whole unstained cells, the morphology of the membranous rearrangements induced in HCV replicon-harbouring cells in conditions close to the living physiological state. All morphological alterations could be reverted by a combination of sofosbuvir/daclatasvir, which are clinically approved antivirals (direct-acting antivirals; DAAs) for HCV infection. Correlatively combining cryo-SXT and 2D synchrotron-based infrared microscopy provides critical information on the chemical nature of specific infection-related structures, which allows specific patterns of the infection process or the DAA-mediated healing process to be distinguished.