SECM stack imaging and full 3D modelling of asymmetric clusters of live cells

Scanning Electrochemical Microscopy (SECM) has shown great strength as a bioanalytical technique for the characterization of single live cell topography, membrane permeability and extracellular reactive oxygen species. However, care must be taken to avoid the presence of adjacent cells in close proximity. Herein, we describe how these clusters of two or more cells may contribute to a combined signal. SECM is commonly coupled with simulated theoretical probe approach curves, allowing surface geometry or electrochemical reactivity to be quantified. Our novel experimental and simulation methodologies including tailored 3D SECM imaging of the live cells are reported here. These 3D modelling techniques allow the generation of 3D x-y-z cell profiles, cell surface maps at an electrode-cell separation, depth scan maps, probe approach curves to any cell spots of interest, and surface topography. The experimental quantification of cell height and topography was performed on cell clusters with an impermeable hydrophilic redox agent, ferrocenecarboxylate, for the deconvolution of these adjacent cell signals. As a proof of concept, experimental and theoretical results were compared to established model outputs. The characterization limits of commonly employed electrode sizes were assessed. Higher complexity cell geometries were explored for the first time, leading to the characterization of these cell clusters with any electrode size. The above developments are versatile, and further demonstrate the strength of SECM as a bioanalytical technique for monitoring cellular homeostasis.