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Microcalcifications serve as diagnostic markers for breast cancer, yet their formation pathway(s) and role in cancer progression are debated due in part to a lack of relevant 3D culture models that allow studying the extent of cellular regulation over mineralization. Previous studies have suggested processes ranging from dystrophic mineralization associated with cell death to bone-like mineral deposition. Here, we evaluated microcalcification formation in 3D multicellular spheroids, generated from non-malignant, pre-cancer, and invasive cell lines from the MCF10A human breast tumor progression series. The spheroids with greater malignancy potential developed necrotic cores, thus recapitulating spatially distinct viable and non-viable areas known to regulate cellular behavior in tumors in vivo . The spatial distribution of the microcalcifications, as well as their compositions, were characterized using nanoCT, electron-microscopy, and X-ray spectroscopy. Apatite microcalcifications were primarily detected within the viable cell regions and their number and size increased with malignancy potential of the spheroids. Levels of alkaline phosphatase decreased with malignancy potential, whereas levels of osteopontin increased. These findings support a mineralization pathway in which cancer cells induce mineralization in a manner that is linked to their malignancy potential, but that is distinct from physiological osteogenic mineralization. [ABSTRACT FROM AUTHOR]

Many important biological questions can be addressed by studying in 3D large volumes of intact, cryo fixed hydrated tissues (⩾10,000 μm 3 ) at high resolution (5–20 nm). This can be achieved using serial FIB milling and block face surface imaging under cryo conditions. Here we demonstrate the unique potential of the cryo-FIB-SEM approach using two extensively studied model systems; sea urchin embryos and the tail fin of zebrafish larvae. We focus in particular on the environment of mineral deposition sites. The cellular organelles, including mitochondria, Golgi, ER, nuclei and nuclear pores are made visible by the image contrast created by differences in surface potential of different biochemical components. Auto segmentation and/or volume rendering of the image stacks and 3D reconstruction of the skeleton and the cellular environment, provides a detailed view of the relative distribution in space of the tissue/cellular components, and thus of their interactions. Simultaneous acquisition of secondary and back-scattered electron images adds additional information. For example, a serial view of the zebrafish tail reveals the presence of electron dense mineral particles inside mitochondrial networks extending more than 20 μm in depth in the block. Large volume imaging using cryo FIB SEM, as demonstrated here, can contribute significantly to the understanding of the structures and functions of diverse biological tissues. [ABSTRACT FROM AUTHOR]

We investigated the manner in which the sea urchin larva takes up calcium from its body cavity into the primary mesenchymal cells (PMCs) that are responsible for spicule formation. We used the membrane-impermeable fluorescent dye calcein and alexa-dextran, with or without a calcium channel inhibitor, and imaged the larvae in vivo with selective-plane illumination microscopy. Both fluorescent molecules are taken up from the body cavity into the PMCs and ectoderm cells, where the two labels are predominantly colocalized in particles, whereas the calcium-binding calcein label is mainly excluded from the endoderm and is concentrated in the spicules. The presence of vesicles and vacuoles inside the PMCs that have openings through the plasma membrane directly to the body cavity was documented using high-resolution cryo-focused ion beam-SEM serial imaging. Some of the vesicles and vacuoles are interconnected to form large networks. We suggest that these vacuolar networks are involved in direct sea water uptake. We conclude that the calcium pathway from the body cavity into cells involves nonspecific endocytosis of sea water with its calcium. [ABSTRACT FROM AUTHOR]

Sea urchin embryos sequester calcium from the sea water. This calcium is deposited in a concentrated form in granule bearing vesicles both in the epithelium and in mesenchymal cells. Here we use in vivo calcein labeling and confocal Raman spectroscopy, as well as cryo-FIB-SEM 3D structural reconstructions, to investigate the processes occurring in the internal cavity of the embryo, the blastocoel. We demonstrate that calcein stained granules are also present in the filopodial network within the blastocoel. Simultaneous fluorescence imaging and Raman spectroscopy show that these granules do contain a calcium mineral. By tracking the movements of these granules, we show that the granules in the epithelium and primary mesenchymal cells barely move, but those in the filopodial network move long distances. We could however not detect any unidirectional movement of the filopodial granules. We also show the presence of mineral containing multivesicular vesicles that also move in the filopodial network. We conclude that the filopodial network is an integral part of the mineral transport process, and possibly also for sequestering calcium and other ions. Although much of the sequestered calcium is deposited in the mineralized skeleton, a significant amount is used for other purposes, and this may be temporarily stored in these membrane-delineated intracellular deposits. [ABSTRACT FROM AUTHOR]

Sea urchin larvae have an endoskeleton consisting of two calcitic spicules. We reconstructed various stages of the formation pathway of calcium carbonate from calcium ions in sea water to mineral deposition and integration into the forming spicules. Monitoring calcium uptake with the fluorescent dye calcein shows that calcium ions first penetrate the embryo and later are deposited intracellularly. Surprisingly, calcium carbonate deposits are distributed widely all over the embryo, including in the primary mesenchyme cells and in the surface epithelial cells. Using cryo-SEM, we show that the intracellular calcium carbonate deposits are contained in vesicles of diameter 0.5–1.5 μm. Using the newly developed airSEM, which allows direct correlation between fluorescence and energy dispersive spectroscopy, we confirmed the presence of solid calcium carbonate in the vesicles. This mineral phase appears as aggregates of 20–30-nm nanospheres, consistent with amorphous calcium carbonate. The aggregates finally are introduced into the spicule compartment, where they integrate into the growing spicule. [ABSTRACT FROM AUTHOR]

Many biogenic minerals are composed of aggregated particles at the nanoscale. These minerals usually form through the transformation of amorphous precursors into single crystals inside a privileged space controlled by the organism. Here, in vitro experiments aimed at understanding the factors responsible for producing such single crystals with aggregated particle texture are presented. Crystallization is achieved by a two-step reaction in which amorphous calcium carbonate (ACC) is first precipitated and then transformed into calcite in small volumes of water and in the presence of additives. The additives used are gel-forming molecules, phosphate ions, and the organic extract from sea urchin embryonic spicules - all are present in various biogenic crystals that grow via the transformation of ACC. Remarkably, this procedure yields faceted single-crystals of calcite that maintain the nanoparticle texture. The crystals grow predominantly by the accretion of ACC nanoparticles, which subsequently crystallize. Gels and phosphate ions stabilize ACC via a different mechanism than sea urchin spicule macromolecules. It is concluded that the unique nanoparticle texture of biogenic minerals results from formation pathways that may differ from one another, but given the appropriate precursor and micro-environment, share a common particle accretion mechanism. [ABSTRACT FROM AUTHOR]

The growth of spontaneously twisted crystals is a common but poorly understood phenomenon. An analysis of the formation of twisted crystals of a metastable benzamide polymorph (form II) crystallizing from highly supersaturated aqueous and ethanol solutions is given here. Benzamide, the first polymorphic molecular crystal reported (1832), would have been the first helicoidal crystal observed had the original authors undertaken an analysis by light microscopy. Polymorphism and twisting frequently concur as they are both associated with high thermodynamic driving forces for crystallization. Optical and electron microscopies as well as electron and powder X‐ray diffraction reveal a complex lamellar structure of benzamide form II needle‐like crystals. The internal stress produced by the overgrowth of lamellae is shown to be able to create a twist moment that is responsible for the observed non‐classical morphologies. [ABSTRACT FROM AUTHOR]

The growth of spontaneously twisted crystals is a common but poorly understood phenomenon. An analysis of the formation of twisted crystals of a metastable benzamide polymorph (form II) crystallizing from highly supersaturated aqueous and ethanol solutions is given here. Benzamide, the first polymorphic molecular crystal reported (1832), would have been the first helicoidal crystal observed had the original authors undertaken an analysis by light microscopy. Polymorphism and twisting frequently concur as they are both associated with high thermodynamic driving forces for crystallization. Optical and electron microscopies as well as electron and powder X‐ray diffraction reveal a complex lamellar structure of benzamide form II needle‐like crystals. The internal stress produced by the overgrowth of lamellae is shown to be able to create a twist moment that is responsible for the observed non‐classical morphologies. [ABSTRACT FROM AUTHOR]

• Characterization of extracellular vesicle (EV) release from human breast cell lines. • SEM shows vesicular structures budding off of membrane tubes driven by HAS3. • Cryo-TEM reveals two EV sub-populations based upon size and structural detail. • EV surface structure is identified as biomarker for MVs compared to exosomes. As interest in the role of extracellular vesicles in cell-to-cell communication has increased, so has the use of microscopy and analytical techniques to assess their formation, release, and morphology. In this study, we evaluate scanning electron microscopy (SEM) and cryo-SEM for characterizing the formation and shedding of vesicles from human breast cell lines, parental and hyaluronan synthase 3-(HAS3)-overexpressing MCF10A cells, grown directly on transmission electron microscopy (TEM) grids. While cells imaged with conventional and cryo-SEM exhibit distinct morphologies due to the sample preparation process for each technique, tubular structures protruding from the cell surfaces were observed with both approaches. For HAS3-MCF10A cells, vesicles were present along the length of membrane protrusions. Once completely shed from the cells, extracellular vesicles were characterized using nanoparticle tracking analysis (NTA) and cryo-TEM. The size distributions obtained by each technique were different not only in the range of vesicles analyzed, but also in the relative proportion of smaller-to-larger vesicles. These differences are attributed to the presence of biological debris in the media, which is difficult to differentiate from vesicles in NTA. Furthermore, we demonstrate that cryo-TEM can be used to distinguish between vesicles based on their respective surface structures, thereby providing a path to differentiating vesicle subpopulations and identifying their size distributions. Our study emphasizes the necessity of pairing several techniques to characterize extracellular vesicles. [ABSTRACT FROM AUTHOR]