We present a new multicolor imaging strategy on a standard fluorescence microscope, where up to four fluorophores with high spectra overlap can be resolved with a single-color optical configuration. Under electrochemical modulation, the fluorophores are regulated between bright states and dim states, with each displaying a distinct fluorescence response pattern. These unique fluorescence-potential profiles enable effective separation of different fluorophores through linear unmixing. We also demonstrated that electrochemical fluorescence switching is readily applicable for multicolor STED imaging. With no modification to the optical setups and easy adaptation to different microscopes, we anticipate that color unmixing based on electrochemical fluorescence switching will provide an easily accessible multicolor imaging pathway for discoveries in diverse fields.

The dataset was collected using an indium tin oxide (ITO)-coated glass coverslip that functioned as both an imaging surface and an electrode. The ITO was connected to a potentiostat with reference and auxiliary electrodes to precisely control the surface potential. The fluorescence intensity of fluorophores was recorded as a function of linearly scanned electrochemical potential. To enable effective modulation, a redox couple (cysteamine and ferricyanide) was introduced into a low-oxygen buffer to mediate the fluorescence modulation across entire fixed and permeabilized cells. Different fluorophores demonstrated distinct fluorescence modulation profiles.

The fluorescence responses of fluorophores were analyzed as a function of the applied potential to extract their characteristic "electrochemical spectra" (EC spectra). The EC spectra were then used as a framework to separate fluorophores in same imaging channel using linear unmixing.