Native diffusion of fluorogenic turn-on dyes accurately report interfacial chemical reaction locations

Single-molecule fluorescence microscopy with "turn-on" dyes that change fluorescent state after a reaction report on the chemistry of interfaces relevant to analytical and bioanalytical chemistry. Paramount to accurately understanding the phenomena at the ultimate detection limit of a single molecule is ensuring fluorophore properties such as diffusion do not obscure the chemical reaction of interest. Here, we develop Monte Carlo simulations of a dye that undergoes reduction to turn-on at the cathode of a corroded iron surface taking into account the diffusion of the dye molecules in a total internal reflection fluorescence (TIRF) excitation volume, location of the cathode, and chemical reactions. We find, somewhat counterintuitively, that a fast diffusion coefficient of D = 10⁸ nm²/s, corresponding to the dye in aqueous solution, accurately reports the location of single reaction sites. The dyes turn on and are present for the acquisition of a single frame allowing for localization before diffusing out of the thin TIRF excitation volume axially. Previously turned-on (i.e., activated) dyes can also randomly hit the surface surrounding the reaction site leading to a uniform increase in the background. Using concentrations that lead to high turnover rates at the reaction site can achieve signal-to-background ratios of  ~100 in our simulation. Therefore, the interplay between diffusion, turn-on reaction rate, and concentration of the dye must be strategically considered to produce accurate images of reaction locations. This work demonstrates that modeling can assist in the design of single-molecule microscopy experiments to understand interfaces related to analytical chemistry such as electrode, nanoparticle, and sensor surfaces.