Real-time nanoscale visualization of cholesterol monohydrate nucleation and growth

Aberrant cholesterol crystallization has implications in the development of numerous pathologies. However, current imaging methods rely on extensive sample preparation and static conditions, unable to capture real-time transformations. This study utilized in-situ graphene liquid cell transmission electron microscopy to capture nanoscale events of cholesterol monohydrate (ChM) nucleation and growth. The results revealed ChM triclinic forms through a combination of non-classical and classical modes, specifically, a modified Stranski - Krastanov mechanism. ChM triclinic nucleates from an amorphous precursor, which grows on triclinic surfaces as an epilayer. These epilayers coalesce into 2D layers formed along a preferred lattice plane, enabling 3D growth. Molecular dynamics simulations revealed that the amorphous to crystalline transition occurs via the selfassembly of small clusters, interconnected by filaments, which regrow into bilayers with exposed polar groups. These superstructures adsorb on the surfaces of crystalline cholesterol, form islands, which spread and form nuclei of a new bilayer. This study underscores the significance of homoepitaxy in ChM growth and may provide additional insights into biologically relevant processes, such as ChM nucleation on lipid droplets. Overall, this study lays the foundation for investigating the mechanisms of ChM growth from solution in realtime and on the nanoscale.