Revealing Natural Photonic Architectures Through the Synergy of Holotomography and Nonlinear Optical Metrology

Historically, photonics has prioritized long-range periodicity to control optical properties, exemplified by ideal photonic crystals. In contrast, biological systems operate under a fundamentally different design principle: short-range-order nanostructures, corrugations, and partially disordered architectures, rather than ideal long-range periodicity, govern optical functionality. Quantitatively linking such structural heterogeneity to optical response requires methods that can resolve both three-dimensional refractive-index distributions and local symmetry-dependent optical signals. This article presents a correlative holotomography (HT)-nonlinear optical (NLO) imaging protocol for thin, weakly scattering biological photonic specimens. The proposed metrology extends from qualitative phase imaging to quantitative HT and from static microscopy to dynamic NLO probing. Neither approach alone is sufficient to decode complex natural photonic architectures. HT provides label-free three-dimensional refractive-index maps, whereas NLO microscopy, particularly polarization-sensitive second-harmonic generation (SHG), reports local non-centrosymmetric organization and anisotropic optical response. Their integration enables a multiscale understanding that links three-dimensional refractive-index landscapes to molecular symmetry, growth dynamics, and energy-dissipation pathways. The protocol describes specimen selection, optical pre-screening, mounting, HT reconstruction, NLO acquisition, cross-modality registration, and quantitative correlative analysis. The workflow is designed to identify spatial associations between RI heterogeneity, RI gradients, and NLO responses. Such correlations provide a basis for hypothesis generation, including photon-phonon-mechanics mechanisms, and motivate future investigations of power-dependent, thermal, mechanical, and time-resolved controls. This method therefore provides a practical framework for studying structure-response relationships in biological photonic architectures while defining boundaries for future investigations of dynamic or adaptive photonic behavior.