Diffusional depth profiling using electrochemiluminescence microscopy.

Abstract

Electrochemiluminescence (ECL) microscopy has emerged as a powerful 2D imaging technique with exceptional spatial resolution, enabling visualization of biological entities, nanomaterials, and multiphase systems. Conventional ECL experiments rely on coreactant concentrations in large excess of the luminophore to generate a bright signal in the vicinity of the electrode. While effective for surface interrogation, this confinement limits resolution in the z-dimension, making 3D imaging challenging. Here, we show that introducing an excess of luminophore enables access to 3D information through diffusion of the emission layer away from the electrode. By tuning the concentration ratio of tris(2,2'-bipyridine)ruthenium(II) ([Ru(bpy)3]2+) to tri-n-propylamine (TPrA), we control emission layer diffusivity. Initially, ECL arises from heterogeneous oxidation of both species at the electrode. As TPrA is depleted, the mechanism shifts to chemiluminescence, where [Ru(bpy)3]3+ oxidizes TPrA, generating a diffusing emissive layer that extends in the z-direction over time. Simulations and scanning electrochemical microscopy capture these diffusion profiles and highlight the role of concentration ratios. We apply this "diffusional depth profiling" to spirulina algae and spheroid cell models, insulating bodies that block light coming from the evolving emissive layer, revealing 3D morphology. This work uncovers the important influence of time and concentration on ECL emission layer expansion for depth profiling micro-to-mesoscale entities across biology and materials science.

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