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RNA viruses, being submicroscopic organisms, have intriguing biological makeups and substantially impact human health. Microscopic methods have been utilized for studying RNA viruses at a variety of scales. In order of observation scale from large to small, fluorescence microscopy, cryo-soft X-ray tomography (cryo-SXT), serial cryo-focused ion beam/scanning electron microscopy (cryo-FIB/SEM) volume imaging, cryo-electron tomography (cryo-ET), and cryo-electron microscopy (cryo-EM) single-particle analysis (SPA) have been employed, enabling researchers to explore the intricate world of RNA viruses, their ultrastructure, dynamics, and interactions with host cells. These methods evolve to be combined to achieve a wide resolution range from atomic to sub-nano resolutions, making correlative microscopy an emerging trend. The developments in microscopic methods provide multi-fold and spatial information, advancing our understanding of viral infections and providing critical tools for developing novel antiviral strategies and rapid responses to emerging viral threats. Full article

Emerging techniques in organelle structural biology have revolutionized our understanding of disease mechanisms and opened new possibilities for developing targeted therapies. In particular, dysfunctions of the Golgi apparatus (GA) have been implicated in a wide range of neurological disorders and cancer, making it a key area of focus in organelle structural biology. The GA plays a crucial role in regulating the transport and modification of proteins and lipids, and dysfunction of this organelle can lead to mislocation and accumulation of proteins and impaired glycosylation, resulting in neurodegenerative diseases such as Parkinson’s Disease and neurodevelopmental disorders (NDDs). Inhibition of vesicular trafficking by α-synuclein may affect dopamine-producing neurons and neuromodulators, while fragmentation and defects within the GA can lead to apoptotic pathways during pathological mechanisms. Additionally, defects and fragmentation of the GA have been implicated in cancer progression, making it a key area of interest for cancer researchers. Advances in imaging technology, such as cryogenic electron tomography, soft-X-ray tomography (SXT), and multiplex correlative light and electron microscopy (CLEM), have enabled high-resolution visualization of the GA and its dysfunctions in neurological diseases and cancer. These techniques provide detailed insight into the structure and function of the GA and have the potential to inform new treatments for diseases associated with GA dysfunction. Recent studies have shown that molecular zippers hold the Golgi membrane together, providing further insight into the mechanisms underlying GA dysfunction in diseases such as Parkinson’s, NDDs, and cancer. Cryo-CLEM and nanobody-assisted tissue immunostaining for volumetric EM (NATIVE) techniques enable high-resolution visualization of the GA and its native environment, aiding in understanding its function in health and disease. In addition, novel techniques such as Optical coherence tomography (OCT) enable rapid, accurate, and high-resolution in vivo imaging of the mouse cortex, providing 3D visualization of cortical microarchitecture using a feature segmentation algorithm. OCT enables label-free, micron-scale 3D imaging of biological tissues’ fine structures with significant depth and a large field of view. A 3D CNS segmentation mask of brain neural networks in a living mouse can be visualized at micron-level resolution using OCT. Overall, the organelle structural biology field, specifically the study of the Golgi apparatus dysfunction in neurological disorders and cancer, has significant implications for developing new therapeutic targets, gene therapy, and drug design. With continued research and advancements in imaging technologies, we can expect to gain a more comprehensive understanding of the underlying mechanisms of GA dysfunction in neurological disorders and cancer, paving the way for innovative new treatments and therapies. Full article

Viruses are obligate parasites that depend on a host cell for replication and survival. Consequently, to fully understand the viral processes involved in infection and replication, it is fundamental to study them in the cellular context. Often, viral infections induce significant changes in the subcellular organization of the host cell due to the formation of viral factories, alteration of cell cytoskeleton and/or budding of newly formed particles. Accurate 3D mapping of organelle reorganization in infected cells can thus provide valuable information for both basic virus research and antiviral drug development. Among the available techniques for 3D cell imaging, cryo–soft X-ray tomography stands out for its large depth of view (allowing for 10 µm thick biological samples to be imaged without further thinning), its resolution (about 50 nm for tomographies, sufficient to detect viral particles), the minimal requirements for sample manipulation (can be used on frozen, unfixed and unstained whole cells) and the potential to be combined with other techniques (i.e., correlative fluorescence microscopy). In this review we describe the fundamentals of cryo–soft X-ray tomography, its sample requirements, its advantages and its limitations. To highlight the potential of this technique, examples of virus research performed at BL09-MISTRAL beamline in ALBA synchrotron are also presented. Full article