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Biophys J, August 2001, p. 1163-1170, Vol. 81, No. 2

Fluorescence Emission Spectral Shift Measurements of Membrane Potential in Single Cells

W. Y. Kao,* C. E. Davis,dagger Y. I. Kim,*§ and J. M. Beach*

Departments of  *Biomedical Engineering and  §Neurology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22906 USA, and  dagger Department of Physiology, Johns Hopkins University, Baltimore, Maryland 21205 USA

Previous measurements of transmembrane potential using the electrochromic probe di-8-ANEPPS have used the excitation spectral shift response by alternating excitation between two wavelengths centered at voltage-sensitive portions of the excitation spectrum and recording at a single wavelength near the peak of the emission spectrum. Recently, the emission spectral shift associated with the change in transmembrane potential has been used for continuous membrane potential monitoring. To characterize this form of the electrochromic response from di-8-ANEPPS, we have obtained fluorescence signals from single cells in response to step changes in transmembrane potentials set with a patch electrode, using single wavelength excitation near the peak of the dye absorption spectrum. Fluorescence changes at two wavelengths near voltage-sensitive portions of the emission spectrum and shifts in the complete emission spectrum were determined for emission from plasma membrane and internal membrane. We found that the fluorescence ratio from either dual-wavelength recordings, or from opposite sides of the emission spectrum, varied linearly with the amplitude of the transmembrane potential step between -80 and +60 mV. Voltage dependence of difference spectra exhibit a crossover point near the peak of the emission spectra with approximately equal gain and loss of fluorescence intensity on each side of the spectrum and equal response amplitude for depolarization and hyperpolarization. These results are consistent with an electrochromic mechanism of action and demonstrate how the emission spectral shift response can be used to measure the transmembrane potential in single cells.

Biophys J, August 2001, p. 1163-1170, Vol. 81, No. 2
© 2001 by the Biophysical Society   0006-3495/01/08/1163/08  $2.00


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