Characterization of di-4-ANEPPS with nano-black lipid membranes

We report a platform based on lateral nano-black lipid membranes (nano-BLMs), where electrical measurements and fluorescence microscopy setup are combined, for the calibration of di-4-ANEPPS, a common voltage sensitive dye (VSD). The advantage of this setup is (1) its flexibility in the choice of lipids and applied voltages, (2) its high stability that enables a high voltage (500 mV) application and long-time measurements and (3) its fluorescence microscopy readout, which can be directly correlated with other fluorescence microscopy experiments using VSDs (e.g. membrane potential measurements in living cells). Using this setup, we observed that the calibration curve of di-4-ANEPPS is strongly dependent on the net electric charge of the lipids. The developed setup can be used to calibrate VSDs in different lipid environments in order to better understand their fundamental voltage-sensing mechanism in the future.