Shell–Core Structural Anisotropy in Starch Granules Probed by Polarization Third-Harmonic Generation Microscopy

Lately the nonlinear optical third-harmonic generation (THG) microscopy is starting to emerge as a laboratory standard for label-free studies in biological samples. In this study, the THG signals produced from corn starch granules are investigated. In particular, the polarization-dependent THG (P-THG) signals emerging from the outer layer (shell) of the starch granules are compared with the P-THG signals originating from their inner portion (core). By rotating the linear polarization of the excitation beam, two distinct P-THG modulation patterns are revealed within single granules, corresponding to their shells and to their structurally different cores. These patterns are analyzed using a theoretical framework that describes THG from an orthorhombic crystal symmetry, characteristic of corn starch. This allows us to extract point-by-point in the granules the ratios of the χ(3) susceptibility tensor elements and the average molecular orientations. Then, the anisotropy ratio (AR = χxxxx(3)/χyyyy(3)) is defined and used as a quantitative descriptor of the local molecular arrangements. Our results show that the shells and cores exhibit distinct AR values, probing the anisotropy in the molecular arrangements between the two regions. This study establishes P-THG as a powerful contrast mechanism for probing structural anisotropy in biological samples beyond conventional THG intensity-only microscopy.