Local Hydration Determines Whether Water Vitrifies or Crystallizes

Understanding how solutes drive water restructuring between crystallization and vitrification remains a central challenge in cryopreservation. Here, we quantitatively compare how two common cryoprotectants, dimethyl sulfoxide (DMSO) and ethylene glycol (EG), modulate water structure at low temperatures. Cryogenic IR spectroscopy of the O-D stretching mode combined with global spectral decomposition reveals a sequential transition from liquid water to vitreous water (VW) to crystalline ice, enabling direct extraction of phase populations as a function of composition and temperature. Both solutes follow this common pathway, with VW emerging as a shared intermediate. The key difference lies in VW stabilization: DMSO induces heterogeneous hydration in which hydrophilic S═O and hydrophobic methyl groups sample distinct local environments, promoting DMSO-rich microdomains that stabilize VW and fully suppress ice formation at 47 wt %. EG maintains a more homogeneous hydration environment, resulting in a smaller VW fraction and partial crystallization. Despite these different macroscopic outcomes, both cryoprotectants employ the same hydrophilic and hydrophobic interactions operating within different molecular architectures. These findings demonstrate that vitrification behavior can be understood at the level of individual molecular architectures, where the spatial arrangement of hydrophilic and hydrophobic motifs shapes local hydration topology, providing a topology-informed basis for rational cryoprotectant design.