Search

Despite extensive research on the nucleation and growth of calcium oxalate (CaOx) crystals, there are still several challenges and unknowns that remain. In particular, the role of trace metal elements in the promotion or inhibition of CaOx crystals is not well understood. In the present study, in situ graphene liquid cell transmission electron microscopy ( in situ GLC TEM) was used to observe real-time, nanoscale transformations of CaOx crystals in the presence of nickel ions (Ni 2+ ). The results showed that Ni 2+ form Ni-water complexes, acting as a shape-directing species, generating a unique morphology and altering growth kinetics. Transient adsorption of Ni-water complexes resulted in a metastable phase formation of calcium oxalate trihydrate. Atomistic molecular dynamics simulations confirmed that Ni 2+ acts as a weak inhibitor which slows down the CaOx crystallization, elucidating that Ni 2+ impacts small-sized CaOx clusters by bringing more water into the clusters. This work highlighted the intricacies behind the effect of Ni 2+ on CaOx biomineralization that were made possible to discern using in situ GLC TEM.

The study of ice nucleation and growth at the nanoscale is of utmost importance in geological and atmospheric sciences. However, existing transmission electron microscopy (TEM) approaches have been unsuccessful in imaging ice formation directly. Herein, we demonstrate how radical scavengers - such as TiO 2 - encased with water in graphene liquid cells (GLCs) facilitate the observation of ice nucleation phenomena at low temperatures. Atomic-resolution imaging reveals the nucleation and growth of cubic ice-phase crystals at close proximity to TiO 2 -water nanointerfaces at low temperatures. Interestingly, both heterogeneously and homogeneously nucleated ice crystals exhibited this cubic phase. Ice crystal nuclei were observed to be more stable at the TiO 2 -water nanointerface, as compared with crystals in the bulk liquid (homogeneous nucleation), suggesting the radical scavenging efficacy of TiO 2 nanoparticles mitigating the electron beam by-products. The present work demonstrates that the use of radical scavengers in GLC TEM shows great promise towards unveiling the nanoscale pathways for ice nucleation and growth dynamic events.

Occlusive thrombosis is a central pathological event in heart attack, stroke, thromboembolism, etc. Therefore, pharmacological thrombolysis or anticoagulation is used for treating these diseases. However, systemic administration of such drugs causes hemorrhagic side-effects. Therefore, there is significant clinical interest in strategies for enhanced drug delivery to clots while minimizing systemic effects. One such strategy is by using drug-carrying nanoparticles surface-decorated with clot-binding ligands. Efforts in this area have focused on binding to singular targets in clots, e.g. platelets, fibrin, collagen, vWF or endothelium. Targeting vWF, collagen or endothelium maybe sub-optimal since in vivo these entities will be rapidly covered by platelets and leukocytes, and thus inaccessible for sufficient nanoparticle binding. In contrast, activated platelets and fibrin are majorly accessible for particle-binding, but their relative distribution in clots is highly heterogeneous. We hypothesized that combination-targeting of 'platelets + fibrin' will render higher clot-binding efficacy of nanoparticles, compared to targeting platelets or fibrin singularly. To test this, we utilized liposomes as model nanoparticles, decorated their surface with platelet-binding peptides (PBP) or fibrin-binding peptides (FBP) or combination (PBP + FBP) at controlled compositions, and evaluated their binding to human blood clots in vitro and in a mouse thrombosis model in vivo. In parallel, we developed a computational model of nanoparticle binding to single versus combination entities in clots. Our studies indicate that combination targeting of 'platelets + fibrin' enhances the clot-anchorage efficacy of nanoparticles while utilizing lower ligand densities, compared to targeting platelets or fibrin only. These findings provide important insights for vascular nanomedicine design.

Ferritin is a protein that regulates the iron ions in humans by storing them in the form of iron oxides. Despite extensive efforts to understand the ferritin iron oxide structures, it is still not clear how ferritin proteins with a distinct light (L) and heavy (H) chain subunit ratio impact the biomineralization process. In situ graphene liquid cell-transmission electron microscopy (GLC-TEM) provides an indispensable platform to study the atomic structure of ferritin mineral cores in their native liquid environment. In this study, we report differences in the iron oxide formation in human spleen ferritins (HSFs) and human heart ferritins (HHFs) using in situ GLC-TEM. Scanning transmission electron microscopy (STEM) along with selected area electron diffraction (SAED) of the mineral core and electron energy loss spectroscopy (EELS) analyses enabled the visualization of morphologies, crystal structures and the chemistry of iron oxide cores in HSFs and HHFs. Our study revealed the presence of metastable ferrihydrite (5Fe2O3·9H2O) as a dominant phase in hydrated HSFs and HHFs, while a stable hematite (α-Fe2O3) phase predominated in non-hydrated HSFs and HHFs. In addition, a higher Fe3+/Fe2+ ratio was found in HHFs in comparison with HSFs. This study provides new understanding on iron-oxide phases that exist in hydrated ferritin proteins from different human organs. Such new insights are needed to map ferritin biomineralization pathways and possible correlations with various iron-related disorders in humans.

Understanding the biomineralization pathways in living biological species is a grand challenge owing to the difficulties in monitoring the mineralization process at sub-nanometer scales. Here, we monitored the nucleation and growth of magnetosome nanoparticles in bacteria and in real time using a transmission electron microscope (TEM). To enable biomineralization within the bacteria, we subcultured magnetotactic bacteria grown in iron-depleted medium and then mixed them with iron-rich medium within graphene liquid cells (GLCs) right before imaging the bacteria under the microscope. Using in situ electron energy loss spectroscopy (EELS), the oxidation state of iron in the biomineralized magnetosome was analysed to be magnetite with trace amount of hematite. The increase of mass density of biomineralized magnetosomes as a function of incubation time indicated that the bacteria maintained their functionality during the in situ TEM imaging. Our results underpin that GLCs enables a new platform to observe biomineralization events in living biological species at unprecedented spatial resolution. Understanding the biomineralization processes in living organisms facilitates the design of biomimetic materials, and will enable a paradigm shift in understanding the evolution of biological species.

While titanium is the metal of choice for most prosthetics and inner body devices due to its superior biocompatibility, the discovery of Ti-containing species in the adjacent tissue as a result of wear and corrosion has been associated with autoimmune diseases and premature implant failures. Here, we utilize the in situ liquid cell transmission electron microscopy (TEM) in a liquid flow holder and graphene liquid cells (GLCs) to investigate, for the first time, the in situ nano-bio interactions between titanium dioxide nanoparticles and biological medium. This imaging and spectroscopy methodology showed the process of formation of an ionic and proteic bio-camouflage surrounding Ti dioxide (anatase) nanoparticles that facilitates their internalization by bone cells. The in situ understanding of the mechanisms of the formation of the bio-camouflage of anatase nanoparticles may contribute to the definition of strategies aimed at the manipulation of these NPs for bone regenerative purposes.