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.