Correlative cryo-soft X-ray tomography and cryo-structured illumination microscopy reveal changes to lysosomes in amyloid-ß-treated neurons.

Protein misfolding is common to neurodegenerative diseases (NDs) including Alzheimer's disease (AD), which is partly characterized by the self-assembly and accumulation of amyloid-beta in the brain. Lysosomes are a critical component of the proteostasis network required to degrade and recycle material from outside and within the cell and impaired proteostatic mechanisms have been implicated in NDs. We have previously established that toxic amyloid-beta oligomers are endocytosed, accumulate in lysosomes, and disrupt the endo-lysosomal system in neurons. Here, we use pioneering correlative cryo-structured illumination microscopy and cryo-soft X-ray tomography imaging techniques to reconstruct 3D cellular architecture in the native state revealing reduced X-ray density in lysosomes and increased carbon dense vesicles in oligomer treated neurons compared with untreated cells. This work provides unprecedented visual information on the changes to neuronal lysosomes inflicted by amyloid beta oligomers using advanced methods in structural cell biology.

Near-Native Imaging of Label-Free Silver Nanoparticles-Triggered 3D Subcellular Ultrastructural Reorganization in Microalgae.

Understanding the spatial orientation of nanoparticles and the corresponding subcellular architecture events favors uncovering fundamental toxic mechanisms and predicting response pathways of organisms toward environmental stressors. Herein, we map the spatial location of label-free citrate-coated Ag nanoparticles (Cit-AgNPs) and the corresponding subcellular reorganization in microalgae by a noninvasive 3D imaging approach, cryo-soft X-ray tomography (cryo-SXT). Cryo-SXT near-natively displays the 3D maps of Cit-AgNPs presenting in rarely identified sites, namely, extracellular polymeric substances (EPS) and the cytoplasm. By comparative 3D morphological assay, we observe that Cit-AgNPs disrupt the cellular ultrastructural homeostasis, triggering a severe malformation of cytoplasmic organelles with energy-producing and stress-regulating functions. AgNPs exposure causes evident disruption of the chloroplast membrane, significant attenuation of the pyrenoid matrix and starch sheath, extreme swelling of starch granules and lipid droplets, and shrinkage of the nucleolus. In accompaniment, the number and volume occupancy of starch granules are significantly increased. Meanwhile, the spatial topology of starch granules extends from the chloroplast to the cytoplasm with a dispersed distribution. Linking the dynamics of the internal structure and the alteration of physiological properties, we derive a comprehensive cytotoxic and response pathway of microalgae exposed to AgNPs. This work provides a perspective for assessing the toxicity at subcellular scales to achieve label-free nanoparticle-caused ultrastructure remodeling of phytoplankton.

Direct Visualization and Restoration of Metallic Ion-Induced Subcellular Ultrastructural Remodeling.

Analysis of cellular ultrastructure dynamics and metal ions' fate can provide insights into the interaction between living organisms and metal ions. Here, we directly visualize the distribution of biogenic metallic aggregates, ion-induced subcellular reorganization, and the corresponding regulation effect in yeast by the near-native 3D imaging approach, cryo-soft X-ray tomography (cryo-SXT). By comparative 3D morphometric assessment, we observe the gold ions disrupting cellular organelle homeostasis, resulting in noticeable distortion and folding of vacuoles, apparent fragmentation of mitochondria, extreme swelling of lipid droplets, and formation of vesicles. The reconstructed 3D architecture of treated yeast demonstrates ∼65% of Au-rich sites in the periplasm, a comprehensive quantitative assessment unobtained by TEM. We also observe some AuNPs in rarely identified subcellular sites, namely, mitochondria and vesicles. Interestingly, the amount of gold deposition is positively correlated with the volume of lipid droplets. Shifting the external starting pH to near-neutral results in the reversion of changes in organelle architectures, boosting the amount of biogenic Au nanoparticles, and increasing cell viability. This study provides a strategy to analyze the metal ions-living organism interaction from subcellular architecture and spatial localization perspectives.

Hydroxyapatite nanoparticles-cell interaction: New approaches to disclose the fate of membrane-bound and internalised nanoparticles.

Hydroxyapatite nanoparticles are popular tools in bone regeneration, but they have also been used for gene delivery and as anticancer drugs. Understanding their mechanism of action, particularly for the latter application, is crucial to predict their toxicity. To this end, we aimed to elucidate the importance of nanoparticle membrane interactions in the cytotoxicity of MG-63 cells using two different types of nanoparticles. In addition, conventional techniques for studying nanoparticle internalisation were evaluated and compared with newer and less exploited approaches. Hydroxyapatite and magnesium-doped hydroxyapatite nanoparticles were used as suspensions or compacted as specular discs. Comparison between cells seeded on the discs and those supplemented with the nanoparticles allowed direct interaction of the cell membrane with the material to be ruled out as the main mechanism of toxicity. In addition, standard techniques such as flow cytometry were inconclusive when used to assess nanoparticles toxicity. Interestingly, the use of intracellular calcium fluorescent probes revealed the presence of a high number of calcium-rich vesicles after nanoparticle supplementation in cell culture. These structures could not be detected by transmission electron microscopy due to their liquid content. However, by using cryo-soft X-ray imaging, which was used to visualise the cellular ultrastructure without further treatment other than vitrification and to quantify the linear absorption coefficient of each organelle, it was possible to identify them as multivesicular bodies, potentially acting as calcium stores. In the study, an advanced state of degradation of the hydroxyapatite and magnesium-doped hydroxyapatite nanoparticles within MG-63 cells was observed. Overall, we demonstrate that the combination of fluorescent calcium probes together with cryo-SXT is an excellent approach to investigate intracellular calcium, especially when found in its soluble form.

Imaging of Virus-Infected Cells with Soft X-ray Tomography.

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.

Monitoring reversion of hepatitis C virus-induced cellular alterations by direct-acting antivirals using cryo soft X-ray tomography and infrared microscopy.

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.

Single Cell Cryo-Soft X-ray Tomography Shows That Each Chlamydia Trachomatis Inclusion Is a Unique Community of Bacteria.

Chlamydiae are strict intracellular pathogens residing within a specialised membrane-bound compartment called the inclusion. Therefore, each infected cell can, be considered as a single entity where bacteria form a community within the inclusion. It remains unclear as to how the population of bacteria within the inclusion influences individual bacterium. The life cycle of Chlamydia involves transitioning between the invasive elementary bodies (EBs) and replicative reticulate bodies (RBs). We have used cryo-soft X-ray tomography to observe individual inclusions, an approach that combines 40 nm spatial resolution and large volume imaging (up to 16 µm). Using semi-automated segmentation pipeline, we considered each inclusion as an individual bacterial niche. Within each inclusion, we identifyed and classified different forms of the bacteria and confirmed the recent finding that RBs have a variety of volumes (small, large and abnormal). We demonstrate that the proportions of these different RB forms depend on the bacterial concentration in the inclusion. We conclude that each inclusion operates as an autonomous community that influences the characteristics of individual bacteria within the inclusion.

Biomimetic inorganic-organic hybrid nanoparticles from magnesium-substituted amorphous calcium phosphate clusters and polyacrylic acid molecules.

Amorphous calcium phosphate (ACP) has been widely found during bone and tooth biomineralization, but the meta-stability and labile nature limit further biomedical applications. The present study found that the chelation of polyacrylic acid (PAA) molecules with Ca2+ ions in Mg-ACP clusters (~2.1 ± 0.5 nm) using a biomineralization strategy produced inorganic-organic Mg-ACP/PAA hybrid nanoparticles with better thermal stability. Mg-ACP/PAA hybrid nanoparticles (~24.0 ± 4.8 nm) were pH-responsive and could be efficiently digested under weak acidic conditions (pH 5.0-5.5). The internalization of assembled Mg-ACP/PAA nanoparticles by MC3T3-E1 cells occurred through endocytosis, indicated by laser scanning confocal microscopy and cryo-soft X-ray tomography. Our results showed that cellular lipid membranes remained intact without pore formation after Mg-ACP/PAA particle penetration. The assembled Mg-ACP/PAA particles could be digested in cell lysosomes within 24 h under weak acidic conditions, thereby indicating the potential to efficiently deliver encapsulated functional molecules. Both the in vitro and in vivo results preliminarily demonstrated good biosafety of the inorganic-organic Mg-ACP/PAA hybrid nanoparticles, which may have potential for biomedical applications.

Four-Dimensional Characterization of the Babesia divergens Asexual Life Cycle, from the Trophozoite to the Multiparasite Stage.

Babesia is an apicomplexan parasite of significance that causes the disease known as babesiosis in domestic and wild animals and in humans worldwide. Babesia infects vertebrate hosts and reproduces asexually by a form of binary fission within erythrocytes/red blood cells (RBCs), yielding a complex pleomorphic population of intraerythrocytic parasites. Seven of them, clearly visible in human RBCs infected with Babesia divergens, are considered the main forms and named single, double, and quadruple trophozoites, paired and double paired pyriforms, tetrad or Maltese Cross, and multiparasite stage. However, these main intraerythrocytic forms coexist with RBCs infected with transient parasite combinations of unclear origin and development. In fact, little is understood about how Babesia builds this complex population during its asexual life cycle. By combining cryo-soft X-ray tomography and video microscopy, main and transitory parasites were characterized in a native whole cellular context and at nanometric resolution. The architecture and kinetics of the parasite population was observed in detail and provide additional data to the previous B. divergens asexual life cycle model that was built on light microscopy. Importantly, the process of multiplication by binary fission, involving budding, was visualized in live parasites for the first time, revealing that fundamental changes in cell shape and continuous rounds of multiplication occur as the parasites go through their asexual multiplication cycle. A four-dimensional asexual life cycle model was built highlighting the origin of several transient morphological forms that, surprisingly, intersperse in a chronological order between one main stage and the next in the cycle.IMPORTANCE Babesiosis is a disease caused by intraerythrocytic Babesia parasites, which possess many clinical features that are similar to those of malaria. This worldwide disease is increasing in frequency and geographical range and has a significant impact on human and animal health. Babesia divergens is one of the species responsible for human and cattle babesiosis causing death unless treated promptly. When B. divergens infects its vertebrate hosts, it reproduces asexually within red blood cells. During its asexual life cycle, B. divergens builds a population of numerous intraerythrocytic (IE) parasites of difficult interpretation. This complex population is largely unexplored, and we have therefore combined three- and four-dimensional imaging techniques to elucidate the origin, architecture, and kinetics of IE parasites. Unveiling the nature of these parasites has provided a vision of the B. divergens asexual cycle in unprecedented detail and is a key step to develop control strategies against babesiosis.

X-ray tomography of cryopreserved human prostate cancer cells: mitochondrial targeting by an organoiridium photosensitiser.

The organoiridium complex Ir[(C,N)2(O,O)] (1) where C, N = 1-phenylisoquinoline and O,O = 2,2,6,6-tetramethyl-3,5-heptanedionate is a promising photosensitiser for Photo-Dynamic Therapy (PDT). 1 is not toxic to cells in the dark. However, irradiation of the compound with one-photon blue or two-photon red light generates high levels of singlet oxygen (1O2) (in Zhang et al. Angew Chem Int Ed Engl 56 (47):14898-14902 https://doi.org/10.1002/anie.201709082,2017), both within cell monolayers and in tumour models. Moreover, photo-excited 1 oxidises key proteins, causing metabolic alterations in cancer cells with potent antiproliferative activity. Here, the tomograms obtained by cryo-Soft X-ray Tomography (cryo-SXT) of human PC3 prostate cancer cells treated with 1, irradiated with blue light, and cryopreserved to maintain them in their native state, reveal that irradiation causes extensive and specific alterations to mitochondria, but not other cellular components. Such new insights into the effect of 1O2 generation during PDT using iridium photosensitisers on cells contribute to a detailed understanding of their cellular mode of action.

Cells Undergo Major Changes in the Quantity of Cytoplasmic Organelles after Uptake of Gold Nanoparticles with Biologically Relevant Surface Coatings.

Here, we use cryo soft X-ray tomography (cryo-SXT), which delivers 3D ultrastructural volumes of intact cells without chemical fixation or staining, to gain insight about nanoparticle uptake for nanomedicine. We initially used dendritic polyglycerol sulfate (dPGS) with potential diagnostic and therapeutic applications in inflammation. Although dPGS-coated gold nanoparticle (dPGS-AuNP) uptake followed a conventional endocytic/degradative pathway in human lung epithelial cell lines (A549), with cryo-SXT, we detected ∼5% of dPGS-AuNPs in the cytoplasm, a level undetectable by confocal light microscopy. We also observed ∼5% of dPGS-AuNPs in a rarely identified subcellular site, namely, lipid droplets, which are important for cellular energy metabolism. Finally, we also found substantial changes in the quantity of cytoplasmic organelles upon dPGS-AuNP uptake over the 1-6 h incubation period; the number of small vesicles and mitochondria significantly increased, and the number of multivesicular bodies and the number and volume of lipid droplets significantly decreased. Although nearly all organelle numbers at 6 h were still significantly different from controls, most appeared to be returning to normal levels. To test for generality, we also examined cells after uptake of gold nanoparticles coated with a different agent, polyethylenimine (PEI), used for nucleic acid delivery. PEI nanoparticles did not enter lipid droplets, but they induced similar, albeit less pronounced, changes in the quantity of cytoplasmic organelles. We confirmed these changes in organelle quantities for both nanoparticle coatings by confocal fluorescence microscopy. We suggest this cytoplasmic remodeling could reflect a more common cellular response to coated gold nanoparticle uptake.

The cellular landscape by cryo soft X-ray tomography.

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.

SuRVoS: Super-Region Volume Segmentation workbench.

Segmentation of biological volumes is a crucial step needed to fully analyse their scientific content. Not having access to convenient tools with which to segment or annotate the data means many biological volumes remain under-utilised. Automatic segmentation of biological volumes is still a very challenging research field, and current methods usually require a large amount of manually-produced training data to deliver a high-quality segmentation. However, the complex appearance of cellular features and the high variance from one sample to another, along with the time-consuming work of manually labelling complete volumes, makes the required training data very scarce or non-existent. Thus, fully automatic approaches are often infeasible for many practical applications. With the aim of unifying the segmentation power of automatic approaches with the user expertise and ability to manually annotate biological samples, we present a new workbench named SuRVoS (Super-Region Volume Segmentation). Within this software, a volume to be segmented is first partitioned into hierarchical segmentation layers (named Super-Regions) and is then interactively segmented with the user's knowledge input in the form of training annotations. SuRVoS first learns from and then extends user inputs to the rest of the volume, while using Super-Regions for quicker and easier segmentation than when using a voxel grid. These benefits are especially noticeable on noisy, low-dose, biological datasets.

Correlative cryo-fluorescence and cryo-soft X-ray tomography of adherent cells at European synchrotrons.

Cryo-soft X-ray tomography (cryo-SXT) is a synchrotron-hosted imaging technique used to analyze the ultrastructure of intact, cryo-prepared cells. Correlation of cryo-fluorescence microscopy and cryo-SXT can be used to localize fluorescent proteins to organelles preserved close to native state. 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. In our recent work, we used cryo-CLXM to characterize GFP-LC3-positive early autophagosomes in nutrient-starved HEK293A cells (Duke et al., 2013). Cup-shaped omegasomes were found to form at "hot-spots" on the endoplasmic reticulum. Furthermore, cryo-SXT image stacks revealed the presence of large complex networks of tubulated mitochondria in the starved cells, which would be challenging to model at this scale and resolution using light or electron microscopy. In this chapter, we detail the cryo-CLXM workflow that we developed and optimized for studying adherent mammalian cells. We show examples of data collected at the three European synchrotrons that currently host cryo-SXT microscopes, and describe how raw cryo-SXT datasets are processed into tomoX stacks, modeled, and correlated with cryo-fluorescence data to identify structures of interest.

Biological applications of cryo-soft X-ray tomography.

X-rays are used for imaging many different types of biological specimen, ranging from live organisms to the individual cells and proteins from which they are made. The level of detail achieved as a result of the imaging varies depending on both the sample and the technique used. One of the most recent technical developments in X-ray imaging is that of the soft X-ray microscope, designed to allow the internal structure of individual biological cells to be explored. With a field of view of ∼10-20 × âˆ¼10-20 µm, a penetration depth of ∼10 µm and a resolution of ∼40 nm(3), the soft X-ray microscope neatly fits between the imaging capabilities of light and electron microscopes.

Imaging endosomes and autophagosomes in whole mammalian cells using correlative cryo-fluorescence and cryo-soft X-ray microscopy (cryo-CLXM).

Cryo-soft X-ray tomography (cryo-SXT) is a powerful imaging technique that can extract ultrastructural information from whole, unstained mammalian cells as close to the living state as possible. Subcellular organelles including the nucleus, the Golgi apparatus and mitochondria have been identified by morphology alone, due to the similarity in contrast to transmission electron micrographs. In this study, we used cryo-SXT to image endosomes and autophagosomes, organelles that are particularly susceptible to chemical fixation artefacts during sample preparation for electron microscopy. We used two approaches to identify these compartments. For early and recycling endosomes, which are accessible to externally-loaded markers, we used an anti-transferrin receptor antibody conjugated to 10nm gold particles. For autophagosomes, which are not accessible to externally-applied markers, we developed a correlative cryo-fluorescence and cryo-SXT workflow (cryo-CLXM) to localise GFP-LC3 and RFP-Atg9. We used a stand-alone cryo-fluorescence stage in the home laboratory to localise the cloned fluorophores, followed by cryo-soft X-ray tomography at the synchrotron to analyse cellular ultrastructure. We mapped the 3D ultrastructure of the endocytic and autophagic structures, and discovered clusters of omegasomes arising from 'hotspots' on the ER. Thus, immunogold markers and cryo-CLXM can be used to analyse cellular processes that are inaccessible using other imaging modalities.