Four‐Dimensional Electrochemiluminescence Microscopy Enables Depth‐Resolved Imaging of Single, Living Spheroids

Three‐dimensional (3D) cell culture systems called spheroids have emerged as a valuable alternative to traditional two‐dimensional cell cultures due to their ability to closely mimic the complexity of in vivo environments. In this study we propose a hybrid “confocal” electrochemiluminescence (ECL) approach that harnesses the optical sectioning ability of confocal microscopy and the high spatial resolution of ECL, overcoming the surface‐confinement barrier of conventional ECL and enabling depth‐resolved mapping of living spheroids up to 150 µm from the electrode while resolving the temporal evolution of ECL. By exploiting the sluggish heterogeneous electron‐transfer kinetics of the 2‐(dibutylamino)ethanol coreactant at ITO electrodes, homogeneous redox events driven by the sequential oxidation of [Ru(bpy) 3 ] 2+ propagate deep into the cellular architecture. Spatiotemporal mapping reveals the evolution of these processes across the spheroid volume, establishing time as an additional dimension for visualizing ECL reaction‐diffusion behavior within living 3D systems over time. We demonstrate that the 3D microenvironment acts as a reaction‐diffusion chamber that confines reactive intermediates, enhancing the signal in internal regions. This method achieves single‐cell resolution and provides a non‐invasive tool to probe internal permeability and redox metabolic states, transitioning ECL from a surface technique to a powerful volumetric imaging modality for tissue‐engineered models.