Cholesterol is essential for human physiology but also plays a key role in pathological conditions such as atherosclerosis and gallstones. Despite its medical significance, fundamental studies on cholesterol crystallization remain limited. In the presentation, we will demonstrate that a binary water-alcohol mixture, serving as a biomimetic lipid surrogate, provides an ideal medium for in situ examination of cholesterol crystallization. Our findings reveal a two-step nucleation process, where clusters first assemble before transitioning into crystalline structures, as observed using oblique illumination microscopy (OIM) and dynamic light scattering (DLS). Additionally, we identify a unique surface dissolution mechanism, distinct from the pathways reported previously. Scattering measurements show that cluster size depends on temperature and water content but remains independent of cholesterol concentration. DLS data further indicate that solvent composition significantly influences induction time and crystal growth rates. In situ atomic force microscopy (AFM) and microfluidics experiments confirm classical nucleation from dislocations and layer-by-layer growth, leading to macrostep formation and eventual growth cessation. Our results suggest that crystal growth occurs via monomer incorporation into kinks through surface diffusion, with diffusion limitations introducing a unique self-inhibition mode of crystallization.
