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We report the discovery that in the presence of chaotropic anions (SCN−) the opening of nanometer biological vesicles at an electrified interface often becomes a two‐step process (around 30 % doublet peaks). We have then used this to independently count molecules in each subvesicular compartment, the halo and protein dense‐core, and the fraction of catecholamine binding to the dense‐core is 68 %. Moreover, we differentiated two distinct populations of large dense‐core vesicles (LDCVs) and quantified their content, which might correspond to immature (43 %) and mature (30 %) LDCVs, to reveal differences in their biogenesis. We speculate this is caused by an increase in the electrostatic attraction between protonated catecholamine and the negatively charged dense‐core following adsorption of SCN−. [ABSTRACT FROM AUTHOR]