Electrochemically Stimulated Autofluorescence of Indium Tin Oxide Surface: The Implications for Fluorescence Microscopy

Electrochemical fluorescence microscopy using indium tin oxide (ITO)-coated glass surfaces is increasingly employed in single-molecule and surface-based imaging. Although ITO typically exhibits low autofluorescence, here we report that under specific conditions, it can induce significant background fluorescent signal. Specifically, when exposed to light with wavelengths below 560 nm, in the presence of oxidative potentials (>0 V) and cysteamine-containing buffer at alkaline pH (8.19–10.75), a localized photoelectrochemical reaction occurs. This reaction leads to the formation of a surface-bound fluorescent species whose intensity increases over time. The rate of this emission growth depends on three key variables: shorter illumination wavelengths accelerate the process; more positive applied potentials enhance emission intensity; and higher laser power further increases the rate. We propose a mechanism in which the ammonium ion form of cysteamine (2-ammoniumethanethiolate) interacts with the ITO surface, bonding to surface oxygen atoms and thiolate groups reacting to form disulfide bridges with other thiolates groups, making cystamine. This effect presents a potential source of background in experiments involving extended ITO illumination, such as electrochemically modulated single-molecule localization microscopy. Researchers using ITO under these conditions should be aware of this phenomenon to avoid misinterpretation of fluorescence signals.