Optical measurement of transmembrane potential in intact cardiac tissues using fluorescent resonance energy transfer

Fluorescent Resonance Energy Transfer [FRET] is the transfer of excitation energy from a donor fluorescent molecule to an acceptor fluorescent molecule. FRET has been developed to achieve fast ratiometric, transmembrane potential sensitive fluorescence changes in single cells. The mechanism relies on the rapid redistribution of hydrophobic fluorescent anions. These anions redistribute from one face of the plasma membrane to the other according to the Nernst equation. A second fluorophore is used to label an extracellular surface and undergoes energy transfer with the membrane bound sensor. During membrane depolarization the FRET is disrupted. This large change in fluorescence allows for imaging of the transmembrane potential. It is the objective of this work to adapt the FRET measurement system for eventual use in intact cardiac tissues. Currently, studies are underway for the individual verification of the functionality of each dye placed in the FRET system. The fluorescent anion experiments have shown the expected decrease in fluorescent intensity with a decrease in transmembrane potential