Dual-wavelength ratiometric fluorescence measurements of membrane potential.
This work shows that the voltage across membranes in two very different preparations, lipid vesicles in suspension and individual HeLa cells under a microscope, is linearly related to the ratio of fluorescence excited from the two wings of the absorption spectrum of a voltage-sensitive dye. The dye di-4-ANEPPS [1-(3-sulfonatopropyl)-4-[beta-[2-(di-n-butylamino)-6-naphthyl] vin yl]pyridinium betaine] is well characterized from earlier investigations and responds via a rapid (less than millisecond) spectral shift to membrane potential changes. The resultant small change in fluorescence intensity monitored at a single wavelength is useful for measurements of temporally well-defined voltage transients such as action potentials. The dual-wavelength approach described in this work extends the usefulness of this fast potentiometric dye by filtering out complex or artifactual changes in fluorescence intensity and providing a voltage-dependent signal that is internally standardized. Thus, rapid measurements of membrane potential are made possible in nonexcitable cells.
Montana
V
V
Department of Physiology, University of Connecticut Health Center, Farmington 06032.
Farkas
D L
DL
Loew
L M
LM
D004553:Electric Conductivity; D005456:Fluorescent Dyes; D006367:HeLa Cells; D006801:Humans; D008055:Lipids / Q000032:analysis; D008564:Membrane Potentials; D011726:Pyridinium Compounds; D013050:Spectrometry, Fluorescence / Q000379:methods
D005456:Fluorescent Dyes; D008055:Lipids; D011726:Pyridinium Compounds; C050019:1-(3-sulfonatopropyl)-4-(beta)(2-(di-n-butylamino)-6-naphthylvinyl)pyridinium betaine