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In this study, a novel application of synchrotron X-ray nanotomography based on high-resolution full-field transmission X-ray microscopy for characterizing the structure and morphology of micrometric hollow polymeric fibers is presented. By employing postimage analysis using an open-source software such as Tomviz and ImageJ, various key parameters in fiber morphology, including diameter, wall thickness, wall thickness distribution, pore size, porosity, and surface roughness, were assessed. Electrospun polycaprolactone fibers with micrometric diameters and submicrometric features with induced porosity via gas dissolution foaming were used to this aim. The acquired synchrotron X-ray nanotomography data were analyzed using two approaches: 3D tomographic reconstruction and 2D radiographic projection-based analysis. The results of the combination of both approaches demonstrate unique capabilities of this technique, not achievable by other available techniques, allowing for a full characterization of the internal and external morphology and structure of the fibers as well as to obtain valuable qualitative insights into the overall fiber structure.
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The combination of near edge X-ray absorption spectroscopy with nanoscale X-ray imaging is a powerful analytical tool for many applications in energy technologies, catalysis, which are critical to combat climate change, as well as microelectronics and life science. Materials from these scientific areas often contain key elements, such as Si, P, S, Y, Zr, Nb, and Mo as well as lanthanides, whose X-ray absorption edges lie in the so-called tender photon energy range 1.5-5.0 keV. Neither conventional grazing incidence grating nor crystal monochromators have high transmission in this energy range, thereby yielding the tender photon energy gap. To close this gap, a monochromator setup based on a multilayer coated blazed plane grating and plane mirror is devised. The measurements show that this novel concept improves the photon flux in the tender X-ray regime by two-orders-of-magnitude enabling previously unattainable laboratory and synchrotron-based studies. This setup is applied to perform nanoscale spectromicroscopy studies. The high photon flux provides sufficient sensitivity to obtain the electronic structure of Mo in platinum-free MoNi4 nanoparticles for electrochemical energy conversion. Additionally, it is shown that the chemical bonding of nano-structures in integrated circuits can be distinguished by the electronic configuration at the Si-K edge.
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The 3D morphology of hierarchically structured electrocatalytic systems is determined based on multi-scale X-ray computed tomography (XCT), and the crystalline structure of electrocatalyst nanoparticles is characterized using transmission electron microscopy (TEM), supported by X-ray diffraction (XRD) and spatially resolved near-edge X-ray absorption fine structure (NEXAFS) studies. The high electrocatalytic efficiency for hydrogen evolution reaction (HER) of a novel transition-metal-based material system - MoNi4 electrocatalysts anchored on MoO2 cuboids aligned on Ni foam (MoNi4/MoO2@Ni) - is based on advantageous crystalline structures and chemical bonding. High-resolution TEM images and selected-area electron diffraction patterns are used to determine the crystalline structures of MoO2 and MoNi4. Multi-scale XCT provides 3D information of the hierarchical morphology of the MoNi4/MoO2@Ni material system nondestructively: Micro-XCT images clearly resolve the Ni foam and the attached needle-like MoO2 micro cuboids. Laboratory nano-XCT shows that the MoO2 micro cuboids with a rectangular cross-section of 0.5 × 1 µm2 and a length of 10-20 µm are vertically arranged on the Ni foam. MoNi4 nanoparticles with a size of 20-100 nm, positioned on single MoO2 cuboids, were imaged using synchrotron radiation nano-XCT. The application of a deep convolutional neural network (CNN) significantly improves the reconstruction quality of the acquired data.
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TiO2 nanoparticles doped with different amounts of Nd3+ (0.5, 1, and 3 wt.%) were synthetized by the sol-gel method, and evaluated as potential temperature nanoprobes using the fluorescence intensity ratio between thermal-sensitive radiative transitions of the Nd3+. XRD characterization identified the anatase phase in all the doped samples. The morphology of the nanoparticles was observed with SEM, TEM and HRTEM microscopies. The relative amount of Nd3+ in TiO2 was obtained by EDXS, and the oxidation state of titanium and neodymium was investigated via XPS and NEXAFS, respectively. Nd3+ was present in all the samples, unlike titanium, where besides Ti4+, a significantly amount of Ti3+ was observed; the relative concentration of Ti3+ increased as the amount of Nd3+ in the TiO2 nanoparticles increased. The photoluminescence of the synthetized nanoparticles was investigated, with excitation wavelengths of 350, 514 and 600 nm. The emission intensity of the broad band that was associated with the presence of defects in the TiO2, increased when the concentration of Nd3+ was increased. Using 600 nm for excitation, the 4F7/2â4I9/2, 4F5/2â4I9/2 and 4F3/2â4I9/2 transitions of Nd3+ ions, centered at 760 nm, 821 nm, and 880 nm, respectively, were observed. Finally, the effect of temperature in the photoluminescence intensity of the synthetized nanoparticles was investigated, with an excitation wavelength of 600 nm. The spectra were collected in the 288-348 K range. For increasing temperatures, the emission intensity of the 4F7/2â4I9/2 and 4F5/2â4I9/2 transitions increased significantly, in contrast to the 4F3/2â4I9/2 transition, in which the intensity emission decreased. The fluorescence intensity ratio between the transitions I821I880=F5/24I49/2F43/2I49/2 and I760I880=F47/2I49/2F43/2I49/2 were used to calculate the relative sensitivity of the sensors. The relative sensitivity was near 3% K-1 for I760I880 and near 1% K-1 for I821I880.
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Nanopartículas , Titanio , Microscopía Electrónica de Transmisión , TemperaturaRESUMEN
Directing nanoparticles to the nucleus by attachment of nuclear localization sequences (NLS) is an aim in many applications. Gold nanoparticles modified with two different NLS were studied while crossing barriers of intact cells, including uptake, endosomal escape, and nuclear translocation. By imaging of the nanoparticles and by characterization of their molecular interactions with surface-enhanced Raman scattering (SERS), it is shown that nuclear translocation strongly depends on the particular incubation conditions. After an 1 h of incubation followed by a 24 h chase time, 14 nm gold particles carrying an adenoviral NLS are localized in endosomes, in the cytoplasm, and in the nucleus of fibroblast cells. In contrast, the cells display no nanoparticles in the cytoplasm or nucleus when continuously incubated with the nanoparticles for 24 h. The ultrastructural and spectroscopic data indicate different processing of NLS-functionalized particles in endosomes compared to unmodified particles. NLS-functionalized nanoparticles form larger intraendosomal aggregates than unmodified gold nanoparticles. SERS spectra of cells with NLS-functionalized gold nanoparticles contain bands assigned to DNA and were clearly different from those with unmodified gold nanoparticles. The different processing in the presence of an NLS is influenced by a continuous exposure of the cells to nanoparticles and an ongoing nanoparticle uptake. This is supported by mass-spectrometry-based quantification that indicates enhanced uptake of NLS-functionalized nanoparticles compared to unmodified particles under the same conditions. The results contribute to the optimization of nanoparticle analysis in cells in a variety of applications, e.g., in theranostics, biotechnology, and bioanalytics.
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Oro , Nanopartículas del Metal , BiotecnologíaRESUMEN
The diatom shell is an example of complex siliceous structure which is a suitable model to demonstrate the process of digging into the third dimension using modern visualization techniques. This paper demonstrates importance of a comprehensive multi-length scale approach to the bio-structures/materials with the usage of state-of-the-art imaging techniques. Imaging of diatoms applying visible light, electron and X-ray microscopy provide a deeper insight into the morphology of their frustules.
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Gold nanostars are a versatile plasmonic nanomaterial with many applications in bioanalysis. Their interactions with animal cells of three different cell lines are studied here at the molecular and ultrastructural level at an early stage of endolysosomal processing. Using the gold nanostars themselves as substrate for surface-enhanced Raman scattering, their protein corona and the molecules in the endolysosomal environment were characterized. Localization, morphology, and size of the nanostar aggregates in the endolysosomal compartment of the cells were probed by cryo soft-X-ray nanotomography. The processing of the nanostars by macrophages of cell line J774 differed greatly from that in the fibroblast cell line 3T3 and in the epithelial cell line HCT-116, and the structure and composition of the biomolecular corona was found to resemble that of spherical gold nanoparticles in the same cells. Data obtained with gold nanostars of varied morphology indicate that the biomolecular interactions at the surface in vivo are influenced by the spike length, with increased interaction with hydrophobic groups of proteins and lipids for longer spike lengths, and independent of the cell line. The results will support optimized nanostar synthesis and delivery for sensing, imaging, and theranostics.
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The Niemann-Pick C2 protein (NPC2) is a sterol transfer protein in the lumen of late endosomes and lysosomes (LE/LYSs). Absence of functional NPC2 leads to endo-lysosomal buildup of cholesterol and other lipids. How NPC2's known capacity to transport cholesterol between model membranes is linked to its function in living cells is not known. Using quantitative live-cell imaging combined with modeling of the efflux kinetics, we show that NPC2-deficient human fibroblasts can export the cholesterol analog dehydroergosterol (DHE) from LE/LYSs. Internalized NPC2 accelerated sterol efflux extensively, accompanied by reallocation of LE/LYSs containing fluorescent NPC2 and DHE to the cell periphery. Using quantitative fluorescence loss in photobleaching of TopFluor-cholesterol (TF-Chol), we estimate a residence time for a rapidly exchanging sterol pool in LE/LYSs localized in close proximity to the plasma membrane (PM), of less than one min and observed non-vesicular sterol exchange between LE/LYSs and the PM. Excess sterol was released from the PM by shedding of cholesterol-rich vesicles. The ultrastructure of such vesicles was analyzed by combined fluorescence and cryo soft X-ray tomography (SXT), revealing that they can contain lysosomal cargo and intraluminal vesicles. Treating cells with apoprotein A1 and with nuclear receptor liver X-receptor (LXR) agonists to upregulate expression of ABC transporters enhanced cholesterol efflux from the PM, at least partly by accelerating vesicle release. We conclude that NPC2 inside LE/LYSs facilitates non-vesicular sterol exchange with the PM for subsequent sterol efflux to acceptor proteins and for shedding of sterol-rich vesicles from the cell surface.
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Membrana Celular/metabolismo , Colesterol/metabolismo , Vesículas Extracelulares/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Células Cultivadas , Humanos , Lisosomas/metabolismoRESUMEN
Gold nanostars are important nanoscopic tools in biophotonics and theranostics. To understand the fate of such nanostructures in the endolysosomal system of living cells as an important processing route in biotechnological approaches, un-labelled, non-targeted gold nanostars synthesized using HEPES buffer were studied in two cell lines. The uptake of the gold nanostructures leads to cell line-dependent intra-endolysosomal agglomeration, which results in a greater enhancement of the local optical fields than those around individual nanostars and near aggregates of spherical gold nanoparticles of the same size. As demonstrated by non-resonant surface-enhanced Raman scattering (SERS) spectra in the presence and absence of aggregation, the spectroscopic signals of molecules are of very similar strength over a wide range of concentrations, which is ideal for label-free vibrational characterization of cells and other complex environments. In 3T3 and HCT-116 cells, SERS data were analyzed together with the properties of the intracellular nanostar agglomerates. Vibrational spectra indicate that the processing of nanostars by cells and their interaction with the surrounding endolysosomal compartment is connected to their morphological properties through differences in the structure and interactions in their intracellular protein corona. Specifically, different intracellular processing was found to result from a different extent of hydrophobic interactions at the pristine gold surface, which varies for nanostars of different spike lengths. The sensitive optical monitoring of surroundings of nanostars and their intracellular processing makes them a very useful tool for optical bionanosensing and therapy.
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Nanopartículas del Metal , Nanoestructuras , Oro , Espectrometría RamanRESUMEN
Understanding the formation of the intracellular protein corona of nanoparticles is essential for a wide range of bio- and nanomedical applications. The innermost layer of the protein corona, the hard corona, directly interacts with the nanoparticle surface, and by shielding the surface, it has a deterministic effect on the intracellular processing of the nanoparticle. Here, we combine a direct qualitative analysis of the hard corona composition of gold nanoparticles with a detailed structural characterization of the molecules in their interaction with the nanoparticle surface and relate both to the effects they have on the ultrastructure of living cells and the processing of the gold nanoparticles. Cells from the cell lines HCT-116 and A549 were incubated with 30 nm citrate-stabilized gold nanoparticles and with their aggregates in different culture media. The combined results of mass spectrometry based proteomics, cryo soft X-ray nanotomography and surface-enhanced Raman scattering experiments together revealed different uptake mechanisms in the two cell lines and distinct levels of induced cellular stress when incubation conditions were varied. The data indicate that the different incubation conditions lead to changes in the nanoparticle processing via different protein-nanoparticle interfacial interactions. Specifically, they suggest that the protein-nanoparticle surface interactions depend mainly on the surface properties of the gold nanoparticles, that is, the ζ-potential and the resulting changes in the hydrophilicity of the nanoparticle surface, and are largely independent of the cell line, the uptake mechanism and intracellular processing, or the extent of the induced cellular stress.
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Nanopartículas del Metal , Nanopartículas , Corona de Proteínas , Oro , Nanopartículas del Metal/toxicidad , Espectrometría Raman , Propiedades de SuperficieRESUMEN
OBJECTIVE: The endothelial glycocalyx covers the luminal surface of the endothelium and plays key roles in vascular function. Despite its biological importance, ideal visualization techniques are lacking. The current study aimed to improve the preservation and subsequent imaging quality of the endothelial glycocalyx. METHODS: In mice, the endothelial glycocalyx was contrasted with a mixture of lanthanum and dysprosium (LaDy). Standard chemical fixation was compared with high-pressure frozen specimens processed with freeze substitution. Also, isolated brain microvessels and cultured endothelial cells were high-pressure frozen and by transmission soft x-rays, imaged under cryogenic conditions. RESULTS: The endothelial glycocalyx was in some tissues significantly more voluminous from chemically fixed specimens compared with high-pressure frozen specimens. LaDy labeling introduced excessive absorption contrast, which impeded glycocalyx measurements in isolated brain microvessels when using transmission soft x-rays. In non-contrasted vessels, the glycocalyx was not resolved. LaDy-contrasted, cultured brain endothelial cells allowed to assess glycocalyx volume in vitro. CONCLUSIONS: Both chemical and cryogenic fixation followed by dehydration lead to substantial collapse of the glycocalyx. Cryogenic fixation without freeze substitution could be a way forward although transmission soft x-ray tomography based solely on amplitude contrast seems unsuitable.
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Criopreservación/métodos , Células Endoteliales/química , Células Endoteliales/ultraestructura , Glicocálix/química , Glicocálix/ultraestructura , Fijación del Tejido/métodos , Animales , Encéfalo/irrigación sanguínea , Encéfalo/citología , Células Cultivadas , Femenino , Substitución por Congelación/métodos , Humanos , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica de Transmisión , Microvasos/citología , Tomografía por Rayos XRESUMEN
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 â¼5% of dPGS-AuNPs in the cytoplasm, a level undetectable by confocal light microscopy. We also observed â¼5% 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.
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Citoplasma/metabolismo , Glicerol/metabolismo , Oro/metabolismo , Nanopartículas del Metal/química , Orgánulos/metabolismo , Polímeros/metabolismo , Sulfatos/metabolismo , Citoplasma/química , Glicerol/química , Oro/química , Humanos , Orgánulos/química , Tamaño de la Partícula , Polímeros/química , Sulfatos/química , Propiedades de Superficie , Tomografía por Rayos X , Células Tumorales CultivadasRESUMEN
The most widely used antimalarial drugs belong to the quinoline family. Their mode of action has not been characterized at the molecular level in vivo. We report the in vivo mode of action of a bromo analog of the drug chloroquine in rapidly frozen Plasmodium falciparum-infected red blood cells. The Plasmodium parasite digests hemoglobin, liberating the heme as a byproduct, toxic to the parasite. It is detoxified by crystallization into inert hemozoin within the parasitic digestive vacuole. By mapping such infected red blood cells with nondestructive X-ray microscopy, we observe that bromoquine caps hemozoin crystals. The measured crystal surface coverage is sufficient to inhibit further hemozoin crystal growth, thereby sabotaging heme detoxification. Moreover, we find that bromoquine accumulates in the digestive vacuole, reaching submillimolar concentration, 1,000-fold more than that of the drug in the culture medium. Such a dramatic increase in bromoquine concentration enhances the drug's efficiency in depriving heme from docking onto the hemozoin crystal surface. Based on direct observation of bromoquine distribution in the digestive vacuole and at its membrane surface, we deduce that the excess bromoquine forms a complex with the remaining heme deprived from crystallization. This complex is driven toward the digestive vacuole membrane, increasing the chances of membrane puncture and spillage of heme into the interior of the parasite.
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Antimaláricos/farmacología , Eritrocitos/parasitología , Malaria Falciparum/parasitología , Plasmodium falciparum/efectos de los fármacos , Quinolinas/farmacología , Cristalización , Eritrocitos/química , Eritrocitos/metabolismo , Hemo/química , Hemo/metabolismo , Hemoproteínas/química , Hemoproteínas/metabolismo , Humanos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/metabolismo , Plasmodium falciparum/fisiologíaRESUMEN
Drugs that influence enzymes of lipid metabolism can cause pathological accumulation of lipids in animal cells. Here, gold nanoparticles, acting as nanosensors that deliver surface-enhanced Raman scattering (SERS) spectra from living cells provide molecular evidence of lipid accumulation in lysosomes after treatment of cultured cells with the three tricyclic antidepressants (TCA) desipramine, amitryptiline, and imipramine. The vibrational spectra elucidate to great detail and with very high sensitivity the composition of the drug-induced lipid accumulations, also observed in fixed samples by electron microscopy and X-ray nanotomography. The nanoprobes show that mostly sphingomyelin is accumulated in the lysosomes but also other lipids, in particular, cholesterol. The observation of sphingomyelin accumulation supports the impairment of the enzyme acid sphingomyelinase. The SERS data were analyzed by random forest based approaches, in particular, by minimal depth variable selection and surrogate minimal depth (SMD), shown here to be particularly useful machine learning tools for the analysis of the lipid signals that contribute only weakly to SERS spectra of cells. SMD is used for the identification of molecular colocalization and interactions of the drug molecules with lipid membranes and for discriminating between the biochemical effects of the three different TCA molecules, in agreement with their different activity. The spectra also indicate that the protein composition is significantly changed in cells treated with the drugs.
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Técnicas Biosensibles , Enzimas/efectos de los fármacos , Producto de la Acumulación de Lípidos , Nanopartículas/química , Esfingomielina Fosfodiesterasa/antagonistas & inhibidores , Colesterol/química , Colesterol/aislamiento & purificación , Inhibidores Enzimáticos/farmacología , Oro/química , Lípidos/química , Lípidos/aislamiento & purificación , Lisosomas/química , Lisosomas/efectos de los fármacos , Nanopartículas del Metal , Espectrometría Raman , Esfingomielina Fosfodiesterasa/química , Esfingomielinas/químicaRESUMEN
A sub-pixel 16 bit charge coupled device camera featuring superresolution for the soft X-ray regime is presented. Superresolution images (SRIs) are reconstructed from a set of 4 × 4 individual low-resolution images that are recorded for different sub-pixel shifts of the detector. SRIs have a 1.3 times higher resolution than individual low-resolution images which is close to the maximum achievable enhancement factor of about 1.5 in the X-ray regime under ideal conditions. To characterize this camera and demonstrate its potential, an X-ray microscope setup is used to image different objects at different photon energies.
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The processing of nanoparticles inside eukaryotic cells is a key step in many wanted and unwanted nano-bio-interactions. In order to understand the effects and functions of the intracellular aggregates that are formed, their properties and their interaction with the biological matrix must be characterized. High quality synchrotron soft X-ray tomography (SXT) data were obtained from cells containing gold nanoparticles that are commonly applied as tools for optical probing or drug delivery. 3D volume rendering of both cellular organelles and the nanoparticle aggregates of different sizes in the intact cells of two cell lines reveals variation in localization, size, shape and density of the intracellular gold nanoaggregates. The dependence of such variation on incubation time and cell type, as well as on the influence of pre-aggregation of primary nanoparticles is shown. The SXT results provide a detailed picture of intracellular aggregation and will improve the design of safe and efficient nanoparticle platforms for biomedical use.
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Structural analysis of biological membranes is important for understanding cell and sub-cellular organelle function as well as their interaction with the surrounding environment. Imaging of whole cells in three dimension at high spatial resolution remains a significant challenge, particularly for thick cells. Cryo-transmission soft X-ray microscopy (cryo-TXM) has recently gained popularity to image, in 3D, intact thick cells (â¼10µm) with details of sub-cellular architecture and organization in near-native state. This paper reports a new tool to segment and quantify structural changes of biological membranes in 3D from cryo-TXM images by tracking an initial 2D contour along the third axis of the microscope, through a multi-scale ridge detection followed by an active contours-based model, with a subsequent refinement along the other two axes. A quantitative metric that assesses the grayscale profiles perpendicular to the membrane surfaces is introduced and shown to be linearly related to the membrane thickness. Our methodology has been validated on synthetic phantoms using realistic microscope properties and structure dimensions, as well as on real cryo-TXM data. Results demonstrate the validity of our algorithms for cryo-TXM data analysis.
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Membrana Celular/ultraestructura , Imagenología Tridimensional/métodos , Microscopía/métodos , Algoritmos , Línea Celular , Criopreservación , Humanos , Imagenología Tridimensional/estadística & datos numéricos , Microscopía/estadística & datos numéricos , Fantasmas de Imagen , Proyectos Piloto , Programas Informáticos , Rayos XRESUMEN
Mast cells play an important role in allergic responses. During activation, these cells undergo degranulation, a process by which various kinds of mediators stored in the granules are released. Granule homeostasis in mast cells has mainly been studied by electron microscopy (EM), where the fine structures of subcellular organelles are partially destroyed during sample preparation. Migration and fusion of granules have not been studied in detail in three dimensions (3D) in unmodified samples. Here, we utilized soft X-ray tomography (SXT) coupled with fluorescence microscopy to study the detailed structures of organelles during mast cell activation. We observed granule fission, granule fusion to plasma membranes, and small vesicles budding from granules. We also detected lipid droplets, which became larger and more numerous as mast cells were activated. We observed dramatic morphological changes of mitochondria in activated mast cells and 3D-reconstruction revealed the highly folded cristae inner membrane, features of functionally active mitochondria. We also observed giant vesicles containing granules, mitochondria, and lipid droplets, which we designated as granule-containing vesicles (GCVs) and verified their presence by EM in samples prepared by cryo-substitution, albeit with a less clear morphology. Thus, our studies using SXT provide significant insights into mast cell activation at the organelle level.
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Anafilaxia/inmunología , Gránulos Citoplasmáticos/ultraestructura , Mastocitos/ultraestructura , Mitocondrias/ultraestructura , Tomografía por Rayos X/métodos , Animales , Degranulación de la Célula , Línea Celular , Espacio Intracelular , Microscopía Electrónica , Nanotecnología , RatasRESUMEN
C60 fullerene crystals may serve as important catalysts for interstellar organic chemistry. To explore this possibility, the electronic structures of free-standing powders of C60 and (C59N)2 azafullerenes are characterized using X-ray microscopy with near-edge X-ray adsorption fine structure (NEXAFS) spectroscopy, closely coupled with density functional theory (DFT) calculations. This is supported with X-ray photoelectron spectroscopy (XPS) measurements and associated core-level shift DFT calculations. We compare the oxygen 1s spectra from oxygen impurities in C60 and C59N, and calculate a range of possible oxidized and hydroxylated structures and associated formation barriers. These results allow us to propose a model for the oxygen present in these samples, notably the importance of water surface adsorption and possible ice formation. Water adsorption on C60 crystal surfaces may prove important for astrobiological studies of interstellar amino acid formation.