In Situ Liquid Cell Transmission Electron Microscopy Studies of Pathological Biomineralization

Pathological biomineralization is a highly dynamic process that underlies a range of diseases, such as kidney stone formation and atherosclerosis. Despite its clinical significance, much of what drives these crystallization events at the nanoscale remains poorly understood. This work leverages in-situ liquid-cell transmission electron microscopy (GLC-TEM) to capture real-time transformations of calcium oxalate and cholesterol, revealing nanoscale pathways that govern stability, growth, and dissolution during crystallization. In-situ GLC-TEM provides an excellent model for confinement during biomineralization and is supported by graphene’s unique properties that enable imaging at the nanoscale. The findings highlight the complexity of pathways observed in real time, such as metastable intermediates, dissolution-precipitation cycles, and structural modifiers, and their roles in influencing mineralization outcomes. These insights could inform new therapeutic strategies for preventing and controlling pathological crystal formation. One of the central observations is that calcium oxalate (CaOx), the primary component of kidney stones, can stabilize or dissolve via a non-classical pathway. Morphologically, it forms core-shell nanoparticles (NPs) that continuously reorganize, dissolving and reprecipitating in response to local conditions. These transformations are driven by nanoscale confinement, structural defects, and hydration levels, which ultimately dictate whether a particle stabilizes into a crystalline phase or dissolves. The presence of porosity and high-reactivity interfaces within these NPs suggests a strong link between nanoarchitecture and stability, which may explain why certain crystal morphologies are more resistant to dissolution and therefore more likely to persist in pathological environments. Beyond the structural evolution of CaOx, this work also uncovers how trace elements act as crystallization modifiers. Specifically, nickel (Ni²⁺) significantly alters CaOx nucleation and growth by forming Ni-water complexes that interfere with phase selection and alter crystallization kinetics. Instead of directly incorporating into the crystal lattice, Ni²⁺ increases water retention within precursor phases, favoring the formation of metastable calcium oxalate trihydrate (COT) over the thermodynamically stable monohydrate (COM). This finding highlights the impact of Ni²⁺ as a shaper of biomineralization pathway selection, opening new approaches for targeted disruption of pathological biomineralization. In-situ GLC TEM was also used to visualize the complexity in the crystallization of cholesterol monohydrate (ChM), a key component of gallstones and arterial plaques. Here, real-time imaging captures a combination of classical and non-classical growth modes, through a modified Stranski-Krastanov mechanism. Specifically, amorphous cholesterol precursors transition into a crystalline phase through self-assembly of small clusters and subsequent creation of bilayers with exposed polar groups. As 2D layers coalesce, further 3D growth occurs via epitaxial layering, with new layers nucleating on pre-existing cholesterol surfaces along preferred lattice planes. This study provides details into how early-stage assemblies influence long-term crystal growth and identifies targets for therapeutic intervention, laying the foundation for real-time nanoscale investigation of ChM in a near-native environment. Taken together, these findings provide next-level insights into the multi-step processes that drive pathological biomineralization in highly dynamic environments on the nanoscale. The identification amorphous intermediates, dissolution-precipitation cycles, and crystallization modifiers underscores the importance of real-time characterization in understanding mineralization behavior. More importantly, these insights create a foundation for rethinking therapeutic strategies, shifting from conventional bulk-phase inhibitors to nanoscale interventions that disrupt crystallization before stable phases can form. The use of advanced imaging techniques presented in this work provides a roadmap for developing targeted approaches to control and prevent pathological biomineralization at its earliest stages.

Author dc:creator

• Lioudmila V. Sorokina (14798127)

• Engineering
• Materials Science

Statement dc:rights

• In Copyright

DOI dc:identifier

https://doi.org/10.25417/uic.32993822.v1

OAI identifier oai:identifier

oai:figshare.com:article/32993822