Search

Matrix vesicles (MVs) are involved in the initial deposition of hydroxyapatite (HAp) during bone mineralization, but their mechanism of action is not yet fully understood.In vitrostudies propose two pathways by which MVs can trigger HAp precipitation: the first is mediated by their enhanced phosphatase activity, and the second suggested to depend on structural components present in MVs to mediate nucleation directly from soluble ions without the requirement of phosphatase activity. However, the relevance of these two pathways for bone mineralization and the relationship between MVs and forming mineral in suchin vitroexperiments remains unclear. Here, we used near-native cryoTEM nanoscale imaging in combination with bulk characterizations to disentangle the content and action of MVs duringin vitromineralization. We show that MVs isolation by conventional ultracentrifugation results in heterogeneous dispersions containing non-vesicular particles, including collagens and proteoglycans, in addition to bilayered vesicles. The separation of phosphatase-enriched MVs from non-vesicular particles and comparative mineralization experiments demonstrated that the ability of MVs to induce fast mineralization, independently of phosphatase activity, depends on the presence of non-vesicular particles. Therefore, we conclude that the primary pathway by which MVs trigger mineralization is through the action of their resident phosphatase enzymes, with the direct mineral nucleation to be a secondary event consequential of their membrane components. Lastly, we observed mineral formation restricted to the extravesicular space or in close proximity to the membrane interface, suggesting that the relationship between MVs and forming mineral is more intricate than previously understood.

Liquid phase electron microscopy has emerged as a powerful technique forin-situobservation of material formation in liquid. However, the interaction of electrons with an aqueous environment leads to the decomposition of water molecules into multiple radicals (radiolysis), which can affect the formation process. Graphene’s ultra-conductive properties have made it an efficient way to mitigate this problem, as it acts as an electron scavenger when used as a window material. However, finding accessible and reliable protocols for the formation of graphene liquid cell (GLC) has been an enormous challenge. Even though loop-assisted transfer has recently been demonstrated as an effective method, the liquid cells are randomly formed and the process of interest is initiated when the GLCs are sealed. This means that the process cannot be imaged at early timepoints, because searching for their location is a time-consuming effort during which the sample is exposed to electrons.Here we report a novel cryogenic/liquid phase correlative light/electron microscopy workflow that addresses some of the limitations of the graphene liquid cells, combining the advantages of fluorescence and electron microscopy. Fluorescence microscopy (FM) is used to locate the GLCs without exposure to electrons and electron microscopy to capture dynamics with nanoscale resolution. This workflow allows imaging to be initiated at a predetermined space and time by vitrifying and thawing at the moment of interest. We demonstrate the workflow by observing non-classical crystallization pathways, but also highlight its potential application in biological systems.

Cryo-correlative light and electron microscopy (cryoCLEM) is a powerful strategy to high resolution imaging in the unperturbed hydrated state, in particular for in combination with cryo-electron tomography (cryoET). In this approach fluorescence microscopy aids localizing the area of interest, and cryogenic focused ion beam/scanning electron microscopy (cryoFIB/SEM) allows preparation of thin cryo-lamellae for cryoET. However, the current method cannot be accurately applied on bulky (3D) samples such as tissues and organoids. 3D cryo-correlative imaging of large volumes is needed to close the resolution gap between cryo-light microscopy and cryoET, placing sub-nanometer observations in a larger biological context. Currently technological hurdles render 3D cryoCLEM an unexplored approach. Here we demonstrate a cryoCLEM workflow for tissues, correlating 3D cryo-Airyscan confocal microscopy with 3D cryoFIB/SEM volume imaging. Accurate correlation is achieved by imprinting a FinderTOP pattern in the sample surface during high pressure freezing, and allows precise targeting for cryoFIB/SEM volume imaging and cryo-lift out for cryoTEM.

Osteons, the main organizational components of human compact bone, are cylindrical structures composed of layers of mineralized collagen fibrils, called lamellae. These lamellae have different orientations, different degrees of organization and different degrees of mineralization where the intrafibrillar and extrafibrillar mineral is intergrown into one continuous network of oriented crystals.While cellular activity is clearly the source of the organic matrix, recent in vitro studies call into question whether the cells are also involved in matrix mineralization, and suggest that this process could be simply driven by the physicochemical conditions in the extracellular matrix.Through the remineralization of demineralized bone matrix, we demonstrate the complete multiscale reconstruction of the 3D structure and composition of the osteon without cellular involvement. We then explore this cell-free in vitro system as a realistic, functional model for the in situ investigation of matrix-controlled mineralization processes. Using a combination of Raman and electron microscopy we show that glycosaminoglycans play a more prominent role than generally assumed in the matrix-mineral interactions, which determine how fast, were, and in which form the mineral is deposited. Our experiments also show that the organization of the collagen is in part a result of its interaction with the developing mineral.

Diatoms are unicellular photosynthetic algae that produce a silica exoskeleton (frustule) which exposes a highly ordered nano to micro scale morphology. In recent years there has been a growing interest in modifying diatom frustules for technological applications. This is achieved by adding non-essential metals to the growth medium of diatoms which in turn modifies morphology, composition, and resulting properties of the frustule. Here, we investigate the frustule formation in diatom Craspedostauros sp., including changes to overall morphology, silica thickness, and composition, in the presence of Al3+ ions at different concentrations. Our results show that in the presence of Al3+ the total silica uptake from the growth medium increases, although a decrease in the growth rate is observed. This leads to a higher inorganic content per diatom resulting in a decreased pore diameter and a thicker frustule as evidenced by electron microscopy. Furthermore, 27Al solid-state NMR, FIB-SEM, and EDS results confirm that Al3+ becomes incorporated into the frustule during the silicification process, thus, improving hydrolysis resistance. This approach may be extended to a broad range of elements and diatom species towards the scalable production of silica materials with tunable hierarchical morphology and chemical composition.

Diatoms are unicellular photosynthetic algae that produce a silica exoskeleton (frustule) which exposes a highly ordered nano to micro scale morphology. In recent years there has been a growing interest in modifying diatom frustules for technological applications. This is achieved by adding non-essential metals to the growth medium of diatoms which in turn modifies morphology, composition, and resulting properties of the frustule. Here, we investigate the frustule formation in diatom Pinnularia sp., including changes to overall morphology, silica thickness, and composition, in the presence of Al