Confocal-Detection Electrochemiluminescence Microscopy for Three-Dimensional Imaging of Electrochemical Reaction Fields

Abstract Electrochemiluminescence (ECL) imaging is an important tool for visualizing electrochemical reactions and is generally performed by wide-field microscopy, which efficiently collects weak emission. The price of this high photon-collection efficiency, however, is that axial information is collapsed into a depth-integrated projection, preventing three-dimensional (3D) visualization. Confocal detection is attractive for 3D ECL imaging because it can distinguish emission from different focal planes, yet its application to ECL has not been systematically explored, mainly because of the limited photon output of ECL reactions. Here, we construct a confocal-detection ECL (CD-ECL) microscopy platform and identify three factors that make CD-ECL difficult: the low efficiency of ECL reactions, which limits photon generation; the optical trade-off between photon collection and spatiotemporal resolution; and the intrinsic instability of ECL reactions, which causes the emission intensity to change during confocal scanning. To address these problems, we first built a shared-objective configuration to compare confocal and wide-field ECL detection. We then optimized the ECL reaction conditions to increase photon generation, examined the trade-off between photon number and spatiotemporal resolution, and developed cyclic volumetric acquisition (CVA) to reduce the influence of temporal intensity variations. Under the optimized conditions, CD-ECL produced 3D images of emission around graphite-felt electrodes and followed the time-dependent evolution of the ECL reaction field. These results establish a confocal optical-detection route to 3D ECL imaging and provide a proof-of-concept basis for depth-dependent and time-resolved measurements under photon-limited conditions.