Introduction. In the chemical synapse, synaptic vesicles (SV) are in charge of neurotransmitter mobilization inside the cell. They are small sub-cellular structures defined by a lipid membrane containing different kinds of proteins including ion channels, transporters, and pumps all mediating ion exchange. This membrane can be considered an electric capacitor. The contribution of different conductances to the vesicular resting potential has been suggested but not described in detail, in part, due the limitations of the traditional techniques to access at this scale. We adapted a method initially used to follow fluctuations in plasma membrane potential, to evaluate changes in the electric potential of these subcellular structures. Material and methods. To monitor changes in the ionic permeabilities of SV we used a hybrid FRET pair: a GFP fused to a vesicular membrane protein and the hydrophobic ion dipicrylamine (DPA) which moves across the membrane in response to a change in the electrical potential, quenching the GFP emission. By this form, a change in membrane polarization is reinterpreted as a variation of the intensity of brightness. We setup the preparation of membrane sheets using a sonifier allowing full access to the intact docked vesicles. Single vesicle imaging was performed using a laser TIRF microscope coupled to a Hamamatsu Orca 12ER CCD camera. Results and Discussion. After loading DPA we perfused solutions with different ionic composition and record fluorescent fluctuations corresponding to the contribution of Na+, K+ and Cl- ions to the vesicular resting potential.