Dual-wavelength ratiometric fluorescence measurements of membrane potential

Biochemistry. 1989 May 30;28(11):4536-9. doi: 10.1021/bi00437a003.


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.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Electric Conductivity
  • Fluorescent Dyes*
  • HeLa Cells
  • Humans
  • Lipids / analysis
  • Membrane Potentials*
  • Pyridinium Compounds*
  • Spectrometry, Fluorescence / methods


  • Fluorescent Dyes
  • Lipids
  • Pyridinium Compounds
  • 1-(3-sulfonatopropyl)-4-(beta)(2-(di-n-butylamino)-6-naphthylvinyl)pyridinium betaine