Region-Resolved Label-Free Raman Profiling of Perturbation Responses in Intestinal Organoids

Abstract In recent years, organoids have emerged as physiologically relevant three-dimensional in vitro models for studying biological processes as they can better recapitulate tissue architecture, spatial organization, and microenvironmental features. However, current organoid studies still rely predominantly on destructive analytical methods and remain limited in spatially resolved detection, broad molecular readout, and sensitive detection of subtle perturbation-associated changes. We established a label-free, spatially resolved, and sensitive Raman-based workflow for evaluating perturbation-associated responses in intestinal organoids. Using irradiation as a controlled perturbation, we observed dose- and time-dependent Raman spectral changes in murine intestinal organoids, with detectable differences even under mild perturbation. These spectral responses were primarily reflected in coordinated variations of the 785 and 1092 cm–1 nucleic-acid-associated Raman bands, showing an early decrease at 2 h and a partial return toward the control level at 24 and 72 h, with further support from corresponding changes in the 1656 cm–1 protein-associated band and the 1583 cm–1 cytochrome c-associated band. Notably, region-resolved Raman analysis revealed spatial heterogeneity, showing that the stem-cell-enriched region exhibited larger perturbation-associated spectral deviations than the differentiated-cell-associated region. Together, these findings support a practical, sensitive, spatially resolved, and resource-efficient workflow for organoid response evaluation, with potential for longitudinal monitoring of dynamic processes.