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Abstract

We previously demonstrated that sub-terahertz irradiation significantly accelerates the formation of hydration structures in protein aqueous solutions under nonequilibrium conditions, immediately after mixing. To monitor this phenomenon, we developed a microwave dielectric measurement technique capable of sensitively detecting time-dependent changes in hydration under sub-terahertz irradiation. This method utilizes the dielectric-dependent modulation of multiply reflected signals in short-path-length samples (Sugiyama et al., Nat. Commun. 14:2825, 2023). However, the physical origin of the observed signal remained unclear, limiting its broader applicability. In this study, we identify the origin as destructive interference between reflections at the probe–sample interface and the bottom surface of the sample container, which arises uniquely under a short-path-length condition satisfying d=λ/4. This finding establishes a clear measurement principle and enables direct evaluation of dielectric changes in biomolecular hydration from raw reflection data without converting to complex permittivity.