Assessment of membrane potential changes using the carbocyanine dye, diS-C₃-(5): synchronous excitation spectroscopy studies

Abstract

The fluorescence of the voltage sensitive dye, diS-C₃-(5), has been analyzed by means of synchronous excitation spectroscopy. Using this rather rare fluorescence technique we have been able to distinguish between the slightly shifted spectra of diS-C₃-(5) fluorescence from cells and from the supernatant. It has been found that diS-C₃-(5) fluorescence in the supernatant can be selectively monitored at λ_exc = 630 nm and λ_em= 650 nm, while the cell associated fluorescence can be observed at λ_exc= 690 nm and λ_em = 710 nm. A modified theory for the diSC₃-(5) fluorescence response to the membrane potential is presented, according to which a linear relationship exists between the logarithmic increment of the dye fluorescence intensity in the supernatant, In I/I°, and the underlying change in the plasma membrane potential, Δψ_p=ψ_p-ψ°_p. The theory has been tested on human myeloid leukemia cells (line ML-1) in which membrane potential changes were induced by valinomycin clamping in various K⁺ gradients. It has been demonstrated that the membrane potential change, Δψ_p,can be measured on an absolute scale.