Figure 6.

Proposed workflow for characterizing the Nernstian behavior of a candidate cationic membrane voltage dye. The working concentration is estimated in steps 1–4; we define it as the maximum dye concentration that does not affect membrane voltage and that yields sufficient amount of signal. 1) The MNC is estimated. 2) The MNC is tested for sufficient signal intensity and the shape of the equilibration profile is inspected. 3) The effect of the dye on V_m is determined by measuring τ_eq at different below-MNC concentrations. 4) Different τ_eq for different dye concentrations indicate that the probe is altering V_m and the working concentration should be reduced and the protocol resumed from step 2. Equal τ_eq indicates that the probe is not altering V_m. 5) Common procedures to test the expected Nernstian dye responses can then be applied, such as the introduction of a ionophore that neutralizes V_m or changes in external pH that induce changes in V_m (21). 6) I_in from the free dye should be separated from the I_in from the bound dye, and [Dye]_in should be calculated from I_in by taking into account the microscope’s points spread function. 7) Because the effects depend on the physiological state of the cell, the procedure should be repeated for every experimental condition. To see this figure in color, go online.