Elementary processes in the eosin-sensitized photooxidation of 3,3′-diaminobenzidine for correlative fluorescence and electron microscopy

The sensitized photopolymerization of 3,3′-diaminobenzidine tetrahydrochloride (DAB) to yield a localized electron-dense precipitate is the basis of a correlative imaging technique, in which fluorescence and transmission electron microscopies are applied to dye-labeled biological samples. In the present work, the eosin (Eo) sensitized photooxidation of DAB has been investigated, as a model system for understanding the complex photochemical mechanism of this imaging process. It was observed that the irradiation with visible light (515 nm) of aqueous solutions of DAB plus Eo triggers a fast photoreaction of DAB, a parallel consumption of dissolved oxygen, and the formation of an optically dense polymer. Time-resolved spectroscopic measurements as a function of solution composition were used to analyze the initial reactive steps of the photoreaction, which are mediated by the Eo lowest excited triplet state (3Eo*). From all these experiments it was concluded that singlet molecular oxygen [O2(1Δg)], produced by the well-known 3Eo* plus O2 reaction, and superoxide radical anion (O2−) are the dominant reactant species in the photoprecipitation reaction. In contrast, in the absence of dissolved oxygen the rate of the photoreaction is only a 15% of the rate determined under aerobic conditions.