Spatiotemporal Interrogation of Single Spheroids Using Multiplexed Nanoplasmonic‐Fluorescence Imaging

Abstract Advances in organoid models, as ex vivo mini‐organs, and the development of screening imaging technologies have continuously driven each other forward. A complete understanding of organoids requires detailed insights into the intertwined intraorganoid and extraorganoid activities and how they change across time and space. This study introduces a multiplexed imaging platform that integrates label‐free nanoplasmonic biosensing with fluorescence microscopy to offer simultaneous monitoring of dynamics occurring within and around arrays of single spheroids with spatiotemporal resolution. The label‐free module employs nanoplasmonic biosensors with extraordinary optical transmission to track biomolecular secretions into the surroundings, while concurrent fluorescence imaging enables structural analysis and viability assessment. To perform multiparametric interrogation of the data from different channels over extended periods, a deep‐learning‐augmented image analysis is incorporated. The platform is applied to tumor spheroids to investigate vascular endothelial growth factor A secretion alongside morphometric changes and viability, showcasing its ability to capture variations in secretion and growth dynamics between untreated and drug‐treated groups. This integrated approach advances comprehensive insights into organoid models and can complement existing technologies to accelerate discoveries in disease modeling and drug development.