Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

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


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 λexc = 630 nm and λem= 650 nm, while the cell associated fluorescence can be observed at λexc= 690 nm and λem = 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, Δψpp-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, Δψp,can be measured on an absolute scale.

This is a preview of subscription content, log in to check access.


  1. Bashford CL (1981) The measurement of membrane potential using optical indicators. Biosci Rep 1:183–196

  2. Bashford CL, Smith JC (1979) The use of optical probes to monitor membrane potential. Methods Enzymol 55:569–586

  3. Chused CL, Wilson HA, Seligmann BE, Tsien RY (1986) Probes for use in the study of leukocyte physiology by flow cytometry. In: Lansing Taylor D et al. (eds) Application of fluorescence in biomedical sciences. Liss, New York, pp 531–544

  4. Gargus JJ, Adelberg EA, Slayman CW (1984) Rapid changes in bidirectional K+ fluxes preceding DMSO-induced granulocytic differentiation of HL-60 human leukemic cells. J Cell Physiol 120:83–90

  5. Hladky SB, Rink TJ (1976) Potential difference and the distribution of ions across the human red blood cell membrane: a study of the mechanism by which the fluorescent cation diS-C3-(5) reports membrane potential. J Physiol 263:287–319

  6. Hoffman JF, Laris PC (1974) Determination of membrane potentials in human and Amphiuma red blood cells by means of a fluorescent probe. J Physiol 239:519–552

  7. lvkov VG, Pechatnikov VA, Ivkova MN (1984) Redistribution of positively charged probes in membrane suspension under the action of transmembrane potential. Gen Physiol Biophys 3:19–30

  8. Ivkova MN, Pechatnikov VA, Ivkov VG (1984) Mechanism of fluorescence response of the probe diS-C3-(5) to transmembrane potential changes. Gen Physiol Biophys 3:97–117

  9. Johnstone RM, Laris PC, Eddy AA (1982) The use of fluorescent dyes to measure membrane potentials: a critique. J Cell Physiol 112:298–301

  10. Kováč L, Bohmerová E, Butko P (1982) Ionophores and intact cells: I. Valinomycin and nigericin act preferentially on mitochondria and not on the plasma membranes of Saccharomyces cerevisiae. Biochim Biophys Acta 721:341–348

  11. Krasne S (1980) Interaction of voltage-sensing dyes with membranes. II Spectrophotometric and electrical correlates of cyanine dye adsorption to membranes. Biophys J 30:441–462

  12. Laris PC, Pershadsingh HA, Johnstone RM (1976) Monitoring membrane potential in Ehrlich ascite tumor cells by meaning of a fluorescent dye. Biochim Biophys Acta 436:475–488

  13. Lloyd JBF (1971) Synchronized excitation of fluorescence emission. Nature 231:64–65

  14. Rink TJ, Montecucco C, Hesketh TR, Tsien RJ (1980) Lymphocyte membrane potential assessed with fluorescent probes.Biochim Biophys Acta 595:15–30

  15. Rottenberg H (1979) The measurements of membrane potential and ΔpH cells, organelles and vesicles. Methods Enzymol 55:547–569

  16. Sims PJ, Waggoner AS, Wang CH, Hoffman JF (1974) Studies on the mechanism by which cyanine dye measure membrane potential in red blood cells and phosphatidylcholine vesicles. Biochemistry 13:3315–3330

  17. Tsien RJ Hladky SB (1978) A quantitative resolution of the spectra of a membrane potential indicator diS-C3-(5) bound to cell components and to red blood cells. J Membr Biol 38:73–97

  18. Vo Dinh T (1981) Synchronous excitation spectroscopy.In: Wehry EC (ed) Modern fluorescence spectroscopy, vol 4. Plenum Press, New York London, p 167

  19. Waggoner AS (1976) Optical probes of membrane potential. J Membr Biol 27:17–334

  20. Waggoner AS (1979) Dye indicators of membrane potential.Ann Rev Biophys Bioeng 8:47–68

  21. Waggoner AS, Wang CH, Tolles RL (1977) Mechanism of potential-dependent light absorption changes in lipid bilayer membranes in the presence of cyanine and oxonol dyes. J Membr Biol 27:317–324

  22. West W, Pearce S (1965) The dimeric state of cyanine dyes. J Phys Chem 69:1894–1903

  23. Wilson HA, Seligmann BE, Chused TM (1985) Voltage sensitive cyanine dye fluorescence signals in lymphocytes. Plasma membrane and mitochondrial components. J Cell Physiol 125:61–71

Download references

Author information

Additional information

Offprint requests to: J. Plasek

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Plášek, J., Hrouda, V. Assessment of membrane potential changes using the carbocyanine dye, diS-C3-(5): synchronous excitation spectroscopy studies. Eur Biophys J 19, 183–188 (1991).

Download citation

Key words

  • Membrane potential
  • Fluorescence
  • diS-C3(5)
  • Synchronous excitation spectroscopy