Laboratory based correlative cryo-soft X-ray tomography and cryo-fluorescence microscopy.

Cryo-soft X-ray tomography is the unique technology that can image whole intact cells in 3D under normal and pathological conditions without labelling or fixation, at high throughput and spatial resolution. The sample preparation is relatively straightforward; requiring just fast freezing of the specimen before transfer to the microscope for imaging. It is also possible to image chemically fixed samples where necessary. The technique can be correlated with cryo fluorescence microscopy to localize fluorescent proteins to organelles within the whole cell volume. Cryo-correlated light and soft X-ray tomography is particularly useful for the study of gross morphological changes brought about by disease or drugs. For example, viral fluorescent tags can be co-localized to sites of viral replication in the soft X-ray volume. In general this approach is extremely useful in the study of complex 3D organelle structure, nanoparticle uptake or in the detection of rare events in the context of whole cell structure. The main challenge of soft X-ray tomography is that the soft X-ray illumination required for imaging has heretofore only been available at a small number of synchrotron labs worldwide. Recently, a compact device with a footprint small enough to fit in a standard laboratory setting has been deployed ("the SXT-100") and is routinely imaging cryo prepared samples addressing a variety of disease and drug research applications. The SXT-100 facilitates greater access to this powerful technique and greatly increases the scope and throughput of potential research projects. Furthermore, the availability of cryo-soft X-ray tomography in the laboratory will accelerate the development of novel correlative and multimodal workflows by integration with light and electron microscope based approaches. It also allows for co-location of this powerful imaging modality at BSL3 labs or other facilities where safety or intellectual property considerations are paramount. Here we describe the compact SXT-100 microscope along with its novel integrated cryo-fluorescence imaging capability.

Progression of herpesvirus infection remodels mitochondrial organization and metabolism.

Viruses target mitochondria to promote their replication, and infection-induced stress during the progression of infection leads to the regulation of antiviral defenses and mitochondrial metabolism which are opposed by counteracting viral factors. The precise structural and functional changes that underlie how mitochondria react to the infection remain largely unclear. Here we show extensive transcriptional remodeling of protein-encoding host genes involved in the respiratory chain, apoptosis, and structural organization of mitochondria as herpes simplex virus type 1 lytic infection proceeds from early to late stages of infection. High-resolution microscopy and interaction analyses unveiled infection-induced emergence of rough, thin, and elongated mitochondria relocalized to the perinuclear area, a significant increase in the number and clustering of endoplasmic reticulum-mitochondria contact sites, and thickening and shortening of mitochondrial cristae. Finally, metabolic analyses demonstrated that reactivation of ATP production is accompanied by increased mitochondrial Ca2+ content and proton leakage as the infection proceeds. Overall, the significant structural and functional changes in the mitochondria triggered by the viral invasion are tightly connected to the progression of the virus infection.

Dehydration as alternative sample preparation for soft X-ray tomography.

Soft X-ray tomography (SXT) is an imaging technique to visualise whole cells without fixation, staining, and sectioning. For SXT imaging, cells are cryopreserved and imaged at cryogenic conditions. Such 'near-to-native' state imaging is in high demand and initiated the development of the laboratory table-top SXT microscope. As many laboratories do not have access to cryogenic equipment, we asked ourselves whether SXT imaging is feasible on dry specimens. This paper shows how the dehydration of cells can be used as an alternative sample preparation to obtain ultrastructure information. We compare different dehydration processes on mouse embryonic fibroblasts in terms of ultrastructural preservation and shrinkage. Based on this analysis, we chose critical point (CPD) dried cells for SXT imaging. In comparison to cryopreserved and air-dried cells, CPD dehydrated cells show high structural integrity although with about 3-7 times higher X-ray absorption for cellular organelles. As the difference in X-ray absorption values between organelles is preserved, 3D anatomy of CPD-dried cells can be segmented and analysed, demonstrating the applicability of CPD-dried sample preparation for SXT imaging. LAY DESCRIPTION: Soft X-ray tomography (SXT) is an imaging technique that allows to see the internal structures of cells without the need for special treatments like fixation or staining. Typically, SXT imaging involves freezing and imaging cells at very low temperatures. However, since many labs lack the necessary equipment, we explored whether SXT imaging could be done on dry samples instead. We compared different dehydration methods and found that critical point drying (CPD) was the most promising for SXT imaging. CPD-dried cells showed high structural integrity, although they absorbed more X-rays than hydrated cells, demonstrating that CPD-dried sample preparation is a viable alternative for SXT imaging.