Electrochemical Dye Switching Assisted Spectral Demixing and 3D STORM Imaging

Super‐resolution microscopy has advanced cellular imaging by enabling nanoscale resolution, while reliable multicolor strategies remain crucial for resolving multiple targets in complex cellular samples. Here, we demonstrate a two‐color imaging strategy using electrochemically controlled stochastic optical reconstruction microscopy (EC‐STORM), which allows precise control over the switching behavior of two cyanine dyes, Alexa 647 and CF680. An oscillating electrochemical potential enables control of emitter density and molecule ON time. This minimizes the spatial and temporal overlap of emitters, enabling effective spectral demixing for two‐color EC‐STORM imaging. We further combined spectral demixing with a biplane detection configuration, enabling additional 3D localization of Alexa 647, where axial coordinates are extracted from the photon‐intensity ratio between the two detection channels instead of point spread function width. This integrated approach offers a practical solution for high‐density multicolor 3D STORM imaging.