Electrochemical fluorescence modulation enables simultaneous multicolour imaging.

Multicolour fluorescence imaging is crucial to simultaneously visualize multiple targets in cells, enabling the study of complicated cellular processes. Common multicolour methods rely on using fluorophores with sufficiently different spectral or lifetime characteristics. Here we present a new multicolour imaging strategy on a standard fluorescence microscope, where up to four fluorophores with high spectral overlap can be resolved using a single-colour optical configuration. We find that under electrochemical modulation, the fluorophores are regulated between the bright and dim states, with each displaying a distinct fluorescence response pattern. These unique fluorescence potential profiles enable the effective separation of different fluorophores through linear unmixing. We also demonstrate that electrochemical fluorescence switching is readily applicable to four-colour STED imaging. With no modification to the optical setups and easy adaptation to different microscopes, we anticipate that colour unmixing based on electrochemical fluorescence switching will provide an easily accessible multicolour imaging pathway for discoveries in diverse fields.